Educational Forum
Recent trends in the nitrer gic nervous system nitrergic P. Roy, G. Venkat Ramana, M.U.R.Naidu, P.Usha Rani
Dept of Clinical Pharmacology and Therapeutics, Nizam’s Institute of Medical Sciences, Panjagutta, Hyderabad. India Received: 18.2.2004 Revised: 1.9.2004 Accepted: 10.10.2004 Correspondence to: M.U.R. Naidu Email:
[email protected]
ABSTRACT The late 20th century witnessed the novel discovery of “Nitrergic” or “Nitroxidergic” innervation of the vascular smooth muscles, their role as a vasodilator in cerebral, ocular and penile vasculature, as well as their reciprocal action to adrenergic vasoconstriction. The identification of this nerve as a postganglionic parasympathetic nerve, the discovery of autonomic efferent nerves where Nitric oxide (NO) is the neurotransmitter (NTM) to blood vessels, its physiological role in the control of smooth muscle tone, and the pharmacological implications of NO have been reviewed. This will aid an in-depth analysis of vascular dysfunctions and the development of strategic pharmacotherapeutic interventions with time. KEY WORDS: Erectile dysfunction, nitrergic neurotransmission, nitroxidergic, neurotransmitter, vasodilatation
Organic nitrates (Nitroglycerine) and sodium nitroprusside release NO which facilitates vascular smooth muscle relaxation and thus, act as NO donors. These NO donors substitute for endogenous NO deficiency, known as “Endothelial Replacement” or “Prodrug” which are effective in different forms of vasospastic disorders.[1] The non-adrenergic non-cholinergic (NANC) inhibitory neurons in various smooth muscles were discovered in the 1970s. Burnstock (1972), Lundberg (1981), Ousman (1988) reported the neurotransmitter role of endogenous relaxants i.e. Substance P, VIP (vasoactive intestinal peptide), CGRP (calcitonin gene related peptide), ATP etc. The role of NO as a neurotransmitter (NTM) is known since 1989. But unlike the then existing definition of neurotransmitters the NO radical is an inorganic, labile, gaseous molecule with low molecular weight.[2] The nerve whose transmitter function depends on the release of NO is called “nitroxidergic” (Toda et al, 1991b; Toda and Okamura, 1991a, 1992c) or “nitrergic” (Rand 1992). However, the NO Nomenclature Committee of the International Union of Pharmacology (chairman: Paul M. Vanhoutte) has chosen “nitrergic” as the official name.[1,3] Neurogenic NO released from the autonomic efferent nerve fibers plays a key role as NTM in blood vessels i.e. it maintains smooth muscle tone of the cerebral and ocular blood vessels leading to their vasodilatation, and smooth muscle relaxation of corpus cavernosum leading to initiation and maintenance of penile erection.[3] Discovery and hypothesis The NANC vasodilator nerves were first discovered in dog cerebral arteries using nicotine. Then various smooth muscle tissues in different animals were tested by different methods,
using agonists and antagonists also. After excluding the effects of other autonomic nerves and NTMs, the hypothesis of NO being a NTM in the vasodilator nerve to blood vessels was made.[3,4] Process of nitric oxide synthesis Electrical impulse (1-20 Hz) or nicotinic stimulation (10-6-4 10 M) generates an action potential in the nerve cell membrane via the pterygopalatine ganglion or presynaptic nicotinic receptor respectively. Thus, depolarization of the nerve terminal leads to opening of the slow channels and calcium (Ca++) influx along the concentration gradient. Further, release of Ca++ from intracellular storage sites results in increased levels of intracellular Ca++. This Ca++ binds with calmodulin (CaM) to form a complex which activates protein kinases. The latter phosphorylates a protein that activates neuronal Nitiric Oxide Synthase (nNOS) in the presence of the calciumcalmodulin (Ca-CaM) complex and co-factors.[3] Meanwhile, L-Arginine is obtained from extracellular space via cationic amino acid transporter (CAT) in the cell membrane, from intracellular sources of protein degradation or by endogenous synthesis i.e. in the liver through the urea cycle, and is also recycled from L-citrulline in neuronal cells and the kidneys. This L-arginine is the substrate for nNOS which helps in the synthesis of NO and L-citrulline in the presence of oxygen. Thereafter, the signal transduction system for smooth muscle relaxation involves activation of soluble guanylyl cyclase (GC) by NO. Activated GC catalyses conversion of GTP to cGMP. cGMP activates protein kinase G while it inhibits Ltype Ca++ channels and thus, calcium entry into cells. Activated protein kinase G ultimately plays a vital role in the reduction of intracellular free Ca++ levels and Ca++ sensitivity. Indian J Pharmacol
| April 2005 | Vol 37 | Issue 2 | 69-76
69
Roy P, et al. Figure 1. Schematic representation of the process of synthesis of nitric oxide. Ca-CaM= Calcium-Calmodulin; FMN= Flavin mononucleotide; FAD= Flavin adenine dinucleotide; BH4= Tetrahydrobiopterin; O2= Oxygen; NO= Nitric oxide; EC= Extracellular; IC= Intracellular; CAT= Cationic amino acid transporter; GC= Guanylyl cyclase; GTP= Guanosine triphosphate; cGMP= Cyclic Guanosine mono phosphate; ω-CT= Omega conotoxin GVIA; ω-7= Omega seven; L-NA= NG-nitro-L-arginine; L-NAME =NW -nitro-L-arginine methyl ester; L-NMMA= NG-monomethyl- L-arginine; ADMA=NG, NG-dimethyl-L-arginine (asymmetric DMA); MB= Methylene blue; ODQ= 1H-(1,2,4) oxadiazolo (4,3-α) quinolin-1-one; OxyHb=oxygenated hemoglobin; Dotted boxes show Inhibitors.
Electrical Impulse
Chemical Stimulus
Generation of action potential and depolarization of nerve terminal ω-CT N-Type Ca++ Channel opens
Non-L, Non-N Type Ca++ channel opens
IC Calmodulin
IC Ca++
Calmidazolium, ω7
Ca-Ca M Complex
Co- Factors : FMN, FAD,NADPH, BH4
EC L-Arg
Active nNOS
CAT
IC L-Arg
O2
NO (nerve terminal)
L-Citrulline
MB, ODQ
L-NMMA, L-NA, L-NAME, ADMA
Protein
PO4 Protein nNOS
Protein Kinases
Active Protein Kinases
NO (vascular smooth muscle)
Soluble GC
Active soluble GC
GTP
cGMP
Active protein kinase G Activates Ca2+- ATPase on plasma membrane, endoplasmic reticulum
Oxy Hb
Protein kinase G
Inhibits
Na+/Ca2+ exchanger
Ca2+-sensitive K+ channels
L-type Ca2+ channel
IC Ca2+, Ca2+ sensitivity smooth muscle relaxation
Thus, Ca++-dependent activation of myosin light-chain kinase is prevented resulting in muscle relaxation (Figures 1, 2).[3,5] The mechanism of release of NO as a NTM is differently modulated by arginine as well as calcium. Calcium influx plays a vital role in nitrergic neurotransmission as evidenced by the attenuation of exocytic NTM release with low Ca++. Besides inhibition of Ca++ influx through N-type channels by ω-conotoxin, effectively reduces nitrergic transmission (Figure 1).[3] The rate of NO production is determined by the enzyme 70
Indian J Pharmacol
| April 2005 | Vol 37 | Issue 2 | 69-76
activity as well as by availability of substrate and cofactors. The isoforms of nitric oxide synthase (NOS) are inducible (iNOS/NOS-2, induced by cytokines) and constitutive (cNOS, activated by Ca++ influx and Ca-CaM complex). iNOS present in inflammatory cells, vascular smooth muscle cells and endothelial cells responds to pathological states. cNOS is present under physiological conditions mainly in the endothelium and neurons known as eNOS (NOS-3) and nNOS (NOS-1) respectively.[6]
Nitrergic nervous system Figure 2. Process of uptake of L-Arginine by the nerve terminal and action of cNOS on substrate L-Arginine for the synthesis of NO. CAT= Cationic amino acid transporter; OTC= Ornithine transcarba-mylase; AL= Arginosuccinate lyase; AS= Arginosuccinate synthetase; O2= Oxygen; NO= Nitric oxide; EC= Extracellular; IC= Intracellular.
L-Ornithine
Urea Arginase EC L-Arg
CAT
IC L-Arg
Carbamoyl phosphate OTC
O2 cNOS
L-Citrulline NO L-Aspartate
L-Fumarate AL
AS L-Arginosuccinate
L-arginine, the only endogenous substrate of NOS, plays an important role in NO production during nervous system development and in disease conditions e.g. stroke, multiple sclerosis, Parkinson’s disease, and HIV dementia. Further, cellular NO production depends on exogenous L-arginine concentration despite theoretical saturation of NOS enzymes with intracellular L-arginine. This phenomenon is known as “Arginine paradox” which may be explained by the following: (1) L-arginine-induced insulin, which has vasodilatory actions. (2) Neither extracellular nor intracellular concentration determines the NOS activity but rather the L-arginine amount transported across the plasma membrane may do so. (3) Endogenous NOS inhibitors (NG, NG-dimethyl-L-arginine, L-citrulline, argininosuccinic acid and agmatine) reduce the enzyme sensitivity to L-arginine. (4) Intracellular L-citrulline is a potent NOS inhibitor so that the cells may need extra L-arginine to compete with L-citrulline inhibition.[6-9] Various agents like oxyhemoglobin, superoxide etc. which affect the release and/or function of NO as a NTM (Figure 1) are discussed in subsequent paragraphs. Confirmation of hypothesis The above hypothesis that NO is a NTM holds good because electrical or nicotinic stimulation of perivascular nerves in animal cerebral arteries causes vasodilation. And this vasodilatation is inhibited by NOS inhibitors like L-NMMA (NG-monomethyl- L-arginine) (which is reversed by L-arginine), L-NA, L-NAME and N-iminoethyl-L-ornithine, Mg2+, Cd2+, calmodulin inhibitors (ω-7, calmidazolium) and on removal of external Ca2+.[10] On removing the endothelium, the actions of NOS inhibitors are abolished. Further, with exogenous application of NO, i.e. acidified NaNO2 or NO donors (nitroglycerine, sodium nitroprusside), dose-dependent cerebral vasodilatation occurs which is abolished by oxy Hb (NO scavenger), methylene blue and [1,2,4]Oxadiazole[4,3-a]quinoxalin-1-one (ODQ) (inhibitors of soluble GC). But stimulation of superfused cerebral artery strips sans endothelium by electrical pulses or nicotine causes release of nitrites and nitrates also.[3]
Confirmation of the role of NO as neurotransmitter (NTM) Antioxidants i.e. pyrogallol, hydroquinone, duroquinone inhibit NO-mediated vasodilation whereas Superoxide dismutase (SOD) enhances such response. In bovine retractor penis muscle and mouse anococcygeus muscle, when the effect of SOD is inhibited by administering Di-ethyl-di-thio-carbamate, the antioxidants inhibit NO response. Thereafter SOD again reverses this inhibition. Thus, endogenous SOD protects neurons from superoxide anions and free radicals and NO is the NTM involved in the vasodilator response.[11,12] Localization of neurons containing NOS The nitrergic neurons are localized by reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) histochemistry, double-label immunohisto-chemistry and immunohistochemistry using antibodies against nNOS, neuropeptideY (NPY) and CGRP.[13,14] The nNOS immunoreactive nerve fibers are located in the proximal and distal portions of the middle cerebral artery (MCA), in the dog. They run irregularly along the arterial walls, at times forming fiber bundles and some times ramifying repeatedly. The thick fibers are located in the outer layer of adventitia while the thin fibers lie towards the lumen. The same is the case with the basilar artery. While fibers to the MCA originate from the ipsilateral pterygopalataline ganglion in dogs, in case of rats they originate from the otic ganglion. Similar nitrergic nerve supply is also found in human cerebral arteries.[15,16] Tracing the origin of nitrergic nerves With electrical stimulation of the sphenopalatine ganglion and facial nerve there is increased cortical blood flow in mammals which is not mediated by ACh (acetylcholine). And this flow decreases in the presence of L-NAME. Further, a nerve action potential generated in the superior salivatory nucleus delivers central information through the geniculate and pterygopalatine ganglion to the cerebral artery and its branches. This regulates their vascular tone under resting and stimulated conditions. Since the histologically confirmed superior salivatory nucleus is a known source of cholinergic preganglionic neuron, only the postganglionic nerve contains both nitrergic and cholinergic neurons.[17] Histochemical studies also confirm the sites releasing NO i.e. from neurons and the endothelium. Moreover, after damaging the pterygopalatine ganglion, the NOS-containing neurons and the vasodilator response to nerve stimulation in the cerebral arterial wall disappears after one week.[18] Pharmacological basis In vitro studies in various mammals with electrical and chemical stimuli produce vascular smooth muscle relaxation in a frequency and concentration-dependent manner, respectively, in partially contracted cerebral arteries. The degree of response varies in different species, cerebral arteries and age groups depending upon the density of NADPH diaphorase/NOS in nerve fibers and the density of nicotinic receptors. Further, the mechanism of vasodilation induced by elec-
Indian J Pharmacol
| April 2005 | Vol 37 | Issue 2 | 69-76
71
Roy P, et al.
trical and nicotinic stimuli seems to be different. This is because the response to electrical stimuli is potentiated by inhibitors of amine transporter while it is abolished by TTX, LNA and ω-CT (N-type Ca++ channel blocker) but not by C6 (hexamethonium). On the other hand the response to nicotine is abolished by C6, other ganglionic blockers and amine transporter inhibitors (e.g. cocaine, tricyclic antidepressants like imipramine and desipramine, propranolol, phentolamine, bretylium, etc.) but is resistant to tetrodotoxin (TTX). While muscarinic receptor activation evokes NO release from nNOS-expressing endothelium, at least two populations of presynaptic nicotinic receptors (TTX-sensitive and TTX-insensitive) are involved in nicotine-induced amine release. The acetylcholine- stimulated nicotinic receptors in nitrergic nerves are responsible for release of NO because atropine completely blocks muscarinic receptors and partially inhibits nicotinic receptors. But cerebral vasodilatation in the presence of high concentration of acetylcholine (ACh) (10-4 M) is resistant to atropine, blunted by cocaine and abolished by C6. So, it is hypothesized that ACh and nicotine probably act on nicotinic receptors in the ganglionic cell membrane increasing ion permeability. This leads to depolarization of cell membrane to a level of firing generated action potential. Further, with electrical stimulation Ca++ influx leads to exocytosis of transmitter vesicles in adrenergic and cholinergic nerves or activates nNOS in nitrergic nerves.[3] Interaction of nitrergic, cholinergic and adrenergic nerves Often a neuronal network shows immunoreactivity to NOS, acetylcholinesterase (AChE) / choline acetyl transferase, VIP, tyrosine hydroxylase and/or NADPH diaphorase. While in some neurons there is coexistence of NOS and VIP, some others have VIP and AChE coexisting. So while NO mainly maintains cerebral arterial tone, VIP etc. modulate the synthesis or release of neuronal NO.[19] ACh and other muscarinic agonists inhibit nitrergic nervemediated cerebral vasodilation which is abolished by muscarinic receptor antagonists (Figure 3). So the former is due to pre-junctional M2 receptor activation by neurogenic ACh . This is not seen in dogs neither in vivo nor in the presence of exogenous NO. Further, the prejunctional α2 adrenoceptors interfere with the synthesis or release of NO but the ßadrenoceptor-cAMP system plays no role. Effects of NO donors and NOS inhibitors on neurogenic NE in the peripheral vasculature in animal studies are controversial. This leads to the suspicion that NO, possibly from nitrergic nerves, prejunctionally inhibits/modulates adrenergic nerve function in some mammalian vasculature. And NOS inhibitors potentiate adrenergic vasoconstriction. This is because nitrergic nerves release NO which acts as physiological antagonist at postjunctional smooth muscle, thus opposing the contractile action of NE. Probably, cGMP is the inhibitory modulator of NE release from adrenergic nerves.[17,20] The response to nitrergic stimulation seems to be similar to that of S-nitrosothiols i.e. S-nitroso-L-cysteine (NOCys), nitrosoglutathione (GSNO) and S-nitroso-L-acetyl-D,L-penicillamine (SNAP). But then the chemical nature of the nitrergic NTM is not identical in all tissues, probably depending on the 72
Indian J Pharmacol
| April 2005 | Vol 37 | Issue 2 | 69-76
Figure 3. Interaction between Nitrergic, Cholinergic and Adrenergic nerves in prejunctional and postjunctional sites. NE=Norepinephrine; α1,α2 =Adrenoceptor; M2 =Muscarinic receptor; NO= Nitric oxide; ACh = Acetylcholine; L-Arg= LArginine; L-Citru= L-Citrulline;PIP2=Phosphatidyl inositol4,5-biphosphate; IP 3=Inositol 1,4,5-triphosphate; DG= Diacyl glycerol;GC=Guanylyl cyclase. Inhibition is indicated by dotted arrow.
ADRENERGIC (nerve terminal)
CHOLINERGIC
NITRERGIC nNOS L-Citru
ACh
L-Arg
NE
M2
α2
NO
α2 ?
M2
NE
NO
α1
SMOOTH MUSCLE
Phospholipase C PIP2
I P3 + DG CONTRACTION
Soluble GC GTP
Cyclic GMP
RELAXATION
local availability of the compounds that form NO adducts. The nitrosothiols like NOCys and GSNO release NO in some tissues.[21,22] Clinical significance The major effects of NO are mediated by the second messenger, cGMP, or by reactive nitrogen derivatives. The latter are produced by interaction of NO with molecular oxygen and superoxide radicals. While some conditions like hypertension, angina and impotency are due to deficiency of NO, some other conditions like circulatory shock, stroke and inflammation are due to excess of NO.[6,7] Role in the central nervous system (CNS) NO plays a major role as a NTM and/or a modulator of ligand-gated receptors in the CNS. It has short- and long-term potentiating effects on excitatory amino acids in brain development and memory. It has a protective role in neuronal degeneration e.g. NOS-2 is implicated in Alzheimer’s disease, multiple sclerosis and Huntington’s disease. But high NO levels can kill cells indiscriminately. 7-Nitroindazole (NOS-1 inhibitor) and L-NAME have significant anti-nociceptive effects. The former also reduces signs of opioid withdrawal in animal models, reduces cerebral blood flow, and reduces size of cerebral infarcts in animal models. Ischaemic cerebral damage is associated with NOS-3 deficiency in mice. NO and cGMP might also play a role in epileptic seizures.[6,7] Nitrergic NO in cerebral vasospasm after subarachnoid hemorrhage (SAH) Late cerebral vasospasm usually follows SAH in one to two weeks when oxyHb is formed from lysis of clots around the
Nitrergic nervous system
cerebral artery. Due to release of vasoconstrictor prostanoids, free radicals i.e. superoxide, hydroxyl radicals and endothelin1, and decreased NO synthesis, the patient sometimes deteriorates with fatal outcome.[3] Nitrergic NO in migraine and cluster headache Primary headache comprises migraine, cluster headache and tension-type headache. Of these the origin of migraine could be due to genetic, hormonal, dietary factors, chemicals, stress, lack of sleep and bright light. These factors seem to trigger vasoconstriction followed by reactionary overfilling of some blood vessels in the brain. Or they stimulate hyper-reactive nerve cells in the brain leading to vasoconstriction followed by vasodilation. Or they may trigger release of pro-inflammatory chemicals in the brain causing vasodilation. The release of NO from blood vessels, perivascular nerve endings or from brain tissue is an important molecular trigger mechanism evidenced during such vasodilation which is also associated with demonstrable calcium channelopathy (pathology of the calcium channels). So while some cases are successfully treated with NOS inhibitors, some others are relieved by flunarizine (Ca++ channel blocker) or sumatriptan (smooth muscle constrictor).[3,23-25] Nitrergic nerves are found in the choroid plexus from the pterygopalatine ganglion, in retinal vessels, in ciliary arteries and in ophthalmic arteries. While the tone of the external ophthalmic artery is maintained by the nitrergic vasodilator nerve as well as the adrenergic vasoconstrictor nerve, the internal one is predominantly innervated by the nitrergic nerve. High NO levels cause destruction of photoreceptor cells in the retina.[26] Nitrergic NO and impaired ocular circulation and glaucoma Application of topical nitroglycerin and other NO donors, nipradilol (new drug liberating NO, also has beta- and alphaadrenoceptor blocking actions) results in fall of intraocular tension, enhanced ocular blood flow and protection of ocular fundus from neuronal damage. Similarly, neuronal and endothelial NO also help in the prevention of atherosclerosis and thrombosis. Thus, the mediation of neurogenic and endothelial NO could also impart neuroprotection to glaucomatous eyes and delay the development of the disease.[3,26,27]
sels in different organs and tissues as well as in different mammals. NO plays a significant role in the central regulation of BP. With NO donors or NOS inhibitors injected microscopically at different sites of the brain i.e. rostral ventro-lateral medulla (attenuates renal nerve sympathetic activity), nucleus tractus solitarius (stimulation of NMDA/ N-Methyl-D-aspartate receptors), and paraventricular nucleus (mediation of GABA/ gamma amino butyric acid) there is a fall in systemic BP. Whereas, with injection in the caudal ventrolateral medulla the opposite response is seen. The NOS-containing nerves are located in the afferent nerves, several nuclei in the brainstem, preganglionic parasympathetic and sympathetic nerves. Neurogenic NO acts mainly as neuromodulator in the CNS (Central nervous system) and as NTM in the peripheral nervous system, particularly in the postganglionic parasympathetic neurons. The sympathetic nerve function, mainly postjunctionally and also prejunctionally is blunted by nerve-derived NO. Apart from the central role of NO in the regulation of BP, NO is also involved in the baro reflex mechanism and control of vagal activity. NO interacts with angiotensin II in the sympathetic nervous system. In rat kidneys, basal release of nNOSderived NO attenuates the proximal fluid uptake, which is probably responsible for the depressor action of NO. Further, this attenuation is abolished by acute renal denervation, thus, confirming the involvement of renal sympathetic nerves. Whether the tubular epithelia are innervated by nitrergic nerves or not is yet to be determined. Moreover, how nNOS- derived NO is involved in the adaptive and compensatory mechanisms under chronic hypertension remains to be elucidated.[3,30] NO donors and cGMP analogs are indicated in hyperlipidemic patients with atherosclerosis who have reduced endogenous NO and have endothelial dysfunction. Further, NO being an antioxidant, it prevents oxidation of low density lipoproteins (LDL) and thus prevents foam cell formation in the vascular wall.[6] Role in the respiratory system In pulmonary vessels endogenous/ inhaled NO reaches pulmonary arterioles and reduces vascular resistance. So NO inhalation is useful in the treatment of PAH (Pulmonary artery hypertension) and RDS (Respiratory distress syndrome).[3,6]
Role in the cardiovascular system (CVS)
Role in the reproductive system
NO is not only a potent vasodilator but also inhibits platelet adhesion and aggregation. It may also play a role in blood coagulation by enhancing fibrinolysis by acting on plasminogen. NO donors protect against ischaemia- and reperfusionmediated endothelial dysfunction. In coronary arteries nitrergic nerves are sparsely distributed and derived from the nodose ganglion. The impaired formation and action of neuronal NO is probably involved in the genesis of coronary small artery vasospasm and facilitates vagus–induced bradycardia via prejunctional modulation of neurotransmission.[6,7,28,29] Nitrergic NO and control of blood pressure (BP) The vascular tone in mammals is reciprocally regulated by sympathetic vasoconstrictor and nitrergic vasodilator nerves. Further, predominant innervations differ among different ves-
In the uterine artery the nitrergic nerves arise from the pelvic paracervical ganglion. Pregnancy and estradiol therapy induce Ca2+ -dependent NOS, augmenting the release of NO from neurons and the endothelium.[31] Nitrergic NO and pre-eclampsia Abnormal cytotrophoblast invasion of spiral arterioles results in decreased uteroplacental perfusion. This causes widespread dysfunction of maternal vascular endothelium. And thus evolves the pathogenesis of pre-eclamptic toxemia (PET) of pregnancy. Long-term inhibition of NOS leads to syndromes like intrauterine growth retardation (IUGR), small-for-date babies etc., similar to those in PET, which respond well to high doses of L-arginine. So inhibition of NOS activity and/or NO Indian J Pharmacol
| April 2005 | Vol 37 | Issue 2 | 69-76
73
Roy P, et al.
function could be a causative factor involved in the development of PET. On the other hand, there can be an increase in NOS activity and NO metabolites in the placenta and plasma of patients with PET and eclampsia, which is directly related to the severity of PET. Thus, these parameters are the “diagnostic indicators” of eclampsia. Increase in these parameters is due to a physiological adaptive response in the patient in order to overcome the increased vascular resistance, and to minimize platelet and leucocyte adhesion to the surface of placental villi. PET could be a maladaptive state wherein the placenta inadequately responds to the demands for altered blood flow in pregnancy. Other than endothelial-derived NO, there is also neurogenic NO and nitrergic innervation of the human uterine vessels and female genital organs.[3,31] Nitrergic NO and penile erection The penis receives three different neuron groups i.e. from the thoracolumbar sympathetic, lumbosacral parasympathetic and lumbosacral somatic pathways. The nitrergic nerves are present in the postganglionic parasympathetic nerves, preganglionic sympathetic and parasympathetic nerves and in the afferent nerves projecting to the sacral parasympathetic nucleus in the penis. The density of such nerve fibers varies with developmental changes, age, hormonal changes and pathological states e.g. it is reduced in diabetics. The corpus cavernosum and penile vessels of both animals and humans are densely innervated by nitrergic nerves. The functional state of the penis depends upon the balance between the contractile (anti-erectile) and dilating (erectile) factors. These factors are determined by the penile vascular tone and smooth muscle contractility of the corpora cavernosa. The increased NO levels in the corpus cavernosum by nerve stimulation leads to penile erection (see Figure 4). So impairment of the nitrergic nerve function leads to erectile dysfunction (ED) e.g. diabetic impotent men. Thus, there is reduced formation of NO metabolites and cGMP and hampered cavernosal relaxation in men with vascular impotence. The use of nitroglycerine ointment/patch have been tried in some such cases.[3,6,32] Advancing age, systemic diseases [diabetes, atherosclerosis, hypertension (HTN), renal failure and endocrine disorders], surgical procedures, trauma, drugs (psychotropics), and psychological problems are some of the causative factors of ED. So, any incoordination between the psychological, humoral, neuronal, and vascular factors lead to ED. Further, organic ED is of vascular, neurogenic and cavernosal type. Impaired neurogenic relaxation of the corpus cavernosum under pathological states could be due to selective degeneration of nitrergic nerves, defective NO formation due to altered expression or activity of NOS, decreased sensitivity to NO, superoxide anion formation and increased extra-cellular inactivation of NO in the cavernosal smooth muscle or expression of dysfunctional soluble GC. There is improvement in nitrergic nerve function in diabetic rats after treatment with antioxidants and aldose reductase inhibitors. In addition to prophylaxis and reliable treatment of causative factors, ED responds well to treatment with PDE V inhibitor (oral sildenafil).[3,32] Concurrent use of sildenafil with nitrates in patients of 74
Indian J Pharmacol
| April 2005 | Vol 37 | Issue 2 | 69-76
Figure 4. Neuronal nitric oxide and penile erection
Stimulation of nitrergic nerve
NO release
Penile artery relaxation
Cavernosal smooth muscle relaxation
Cavernosal blood pooling
Expansion of cavernous tissue
Arterial flow Cavernosal capacitance
Compression of plexus of subtunical venules against tunica albuginea Venous outflow
Intracavernous pressure Penile erection
angina sometimes causes severe hypotension and myocardial infarction. In such cases a 6 h gap between the administration of both drugs is essential. Sildenafil may also affect color vision i.e. blue-green discrimination. Moreover, in patients with nerve injury and loss of potency or in those lacking libido, when ED does not respond to sildenafil, treatment with alprostadil, a PGE1 analog, or with phentolamine injection into the cavernosa or with oral apomorphine (investigational drug which acts by releasing dopamine in the CNS) may prove useful. Patients developing adverse effects with sildenafil may be treated with newer PDE V inhibitors like tadalafil and vardenafil.[6] Ageing-related attenuated erectile function also responds successfully to long-term oral L-Arginine. Further, in vivo gene transfer of eNOS, alone or with PDE-V inhibitor, improves erectile response to cavernosal nerve stimulation.[3,32] Role in the immune system Increased NO secretion by activated macrophages and neutrophils is a vital cytotoxic agent in the human immune defense system. Tumor cell lysis by NO is either due to DNA damage or inhibition of tumor cells’ ribonucleotide reductase or due to inactivation of cytoplasmic enzymes and proteins. L-arginine (NO precursor) enhances the natural killer (NK) and lymphokine-activated killer (LAK) cell activity. It is helpful in immuno-suppressive states like AIDS and cancer. NO plays a role in tissue damage due to septic shock, heat stroke, ulcerative colitis, cerebral malaria and autoimmune diabetes mellitus. Administration of NOS inhibitors proves beneficial in such cases.[6,7] Nitrergic NO in acupuncture, axon reflex and neurogenic inflammation NO facilitates CGRP release from afferent neurons or probably acts as second messenger to CGRP in rat skin because NO plays a role in rat paw edema formation. But in larger mammals, NO from efferent vasodilator nerves and CGRP from sensory nerves mediate axon reflex, acupuncture response, inflammatory and immune response in the skin. Vasodilator response of NO is not seen here.[3]
Nitrergic nervous system
Role of NO in hypercapnea, hypoxia, hypothermia and autoregulation The vasodilator response to NO is enhanced by hypercapnic acidosis, possibly due to reduced degradation of NO in animals.[33] On the other hand, hypoxia causes cerebral vasodilation both by acting on non-vascular cerebral elements as well as by vascular effect in the endothelium, smooth muscles and perivascular ner ves. So there is production of many vasodilating metabolites (potassium, H+, prostaglandin, adenosine, NO, cytochrome P450 epoxygynase etc.), neuronal release of excitatory amino acids (leading to cerebral metabolism), direct vasodilator effect on cerebral arteries, decreased calcium influx, production of endothelium-derived cycloxygenase (leading to the activation of ATP-sensitive K+ channels) and activation of ATP- and Ca++- sensitive K+ channels.[34] The effects of hypothermia on neurogenic vascular relaxation in normoxia and hypoxia is mediated by neurogenic NO. But in some isolated arterial studies, there is augmentation of vasodilator nerve function during hypercapnea, whereas in others this function is impaired due to hypoxia. Of course, the latter is prevented by hypothermia, amiloride (Na+-H+ exchange inhibitor) or by extracellular acidosis. Hypothermia leads to intracellular acidosis which can be beneficial by increasing blood supply to the ischemic sites of the brain.[35] Autoregulation is defined as the constant maintenance of blood flow to the vital organs in spite of rapid changes in BP. The myogenic theory of the smooth muscle cells which sense and evoke the response partly explains this mechanism. NO plays an important role as an endothelium derived relaxing factor (EDRF) and as NTM of vasodilator nerve.[36] Pharmacological implications of neurogenic NO PDE-V (Phosphodiesterase type V) Inhibitors These inhibit a cGMP-degrading enzyme (PDE-V) e.g. sildenafil, tadalafil, vardenafil, an effective first line treatment for ED. A few cardiovascular side effects have been reported for which safety of its long-term use is still debatable. As observed in rats, if in humans also, the increased rate of cGMP hydrolysis by PDE-V proves a major contributing factor to the impairment of NO-mediated cerebral vasodilation following SAH, then PDE V inhibitors can be used for prophylaxis of cerebral vasospasm also.[3,6,37] Oral PDE-V inhibitor alone or with inhaled iloprost (stable analog of prostacyclin) is effective in the treatment of pulmonary hypertension. Dosage required is inadequate to lower systemic BP. Sildenafil also ameliorates the effects of inhaled NO withdrawal. PDE-V inhibition improves circulatory disturbances in the brain, eyes, and uterus by vasodilator nerve functions.[3,37] Free radical scavenger Edaravone (3-methyl-1-phenyl-2-pyrazoline-5-one) is effective in the prevention and treatment of symptoms in patients with cerebral ischemia. Probably, it scavenges ischemiagenerated hydroxyl radicals and thereby prevents damage to the brain and circulating system by restoring the actions of neurogenic and endothelial NO.[3]
Alpha2 –adrenoceptor antagonists and antimuscarinic agents In some cerebral, retinal, ciliary and uterine arteries and corpus cavernosum, where nitrergic innervation predominates over adrenergic system, the alpha2–adrenoceptor antagonists potentiate the release of NO and NO function. Non-selective and M2-selective muscarinic receptor antagonists also augment the response to nitrergic nerve stimulation.[17] Neuronal NOS inhibitors The development of nNOS, eNOS, and iNOS selective inhibitors has been evolving over time. In spite of their limited role in cardiovascular diseases, nNOS-derived NO helps in the development of a hyperdynamic circulation consisting of low BP, low systemic vascular resistance and high cardiac output in liver cirrhosis. Keeping NOS inhibitor in view, newer line of treatment of migraine, prevention of nerve cell damage in the brain and retina following ischemic insult, and action on tumor vascular resistance, besides others, are being evaluated.[3] Agents which suppress endogenous NOS inhibitors Accumulated ADMA impairs NO synthesis which forms the basis for vascular and immune dysfunction in CRF patients. Increased plasma levels of endogenous NOS inhibitors are seen in cases of renal failure, DM, HTN, and hypercholesterolemia. While results with administration of exogenous ADMA is controversial, a novel protein inhibitor of nNOS (PIN) shows some promising outcome in the regulation of renal function. Human studies are essential to confirm the same.[3] Conclusion The nitrergic vasodilator nerves contribute mainly to regulate the tone in the cerebral blood vessels than in the peripheral ones. This stresses the fact that under physiological conditions in vivo, when cerebral arteries are constricted, blood flow is controlled mainly by the degree of vasodilation. Thus, the maintained vascular tone in the brain may be due to transmural pressure, circulating vasoconstrictors and weak adrenergic nerve activity. In contrast, in the peripheral arteries except for the coronary artery, it is well understood that the vascular tone is regulated mainly by adrenergic nerve activity. Vasodilation is induced mainly by a decrease in adrenergic nerve activity and an increase in nitrergic activity. Moreover about two-thirds of the vasodilation is due to neuronal NO that is activated by continuing impulses from the vasomotor center. The remaining one-third may be due to NO from the endothelium.[38] In the existing classification of the autonomic nervous system, the parasympathetic nervous system needs to include the nitrergic nervous system as well. More research is required to confirm the etiopathogenesis of diseases associated with depressed or excess NO formation and develop suitable pharmacotherapy for their treatment. References 1. 2.
Moncada S, Higgs A, Furchgott RF.XIV International Union of Pharmacology nomenclature in nitric oxide research. Pharmacol Rev 1997;49:137-42. Walker HA,McGing E,Fisher I,Boger RH,Bode-Boger SM,Jackson G, et al. Endothelium-dependent vasodilatation is independent of the plasma L-arginine/ Indian J Pharmacol
| April 2005 | Vol 37 | Issue 2 | 69-76
75
Roy P, et al.
3. 4. 5. 6. 7. 8. 9. 10. 11.
12. 13. 14. 15. 16. 17. 18. 19. 20.
ADMA ratio in men with stable angina: lack of effect of oral L-arginine on endothelial function, oxidative stress and exercise performance. J Am Coll Cardiol 2001;38:499-505. Toda N, Okamura T. The pharmacology of nitric oxide in the peripheral nervous system of blood vessels. Pharmacol Rev 2003;55:271-324. Esplugues JV. NO as a signalling molecule in the nervous system. Br J Pharmacol 2002;135:1079-95. Wiesinger H. Arginine metabolism and the synthesis of nitric oxide in the nervous system. Prog Neurobiol 2001;64:365-91. Thomas G, Ramwell PW. Nitric oxide, donors and inhibitors. In: Bertram G Katzung, editor. Basic and Clinical Pharmacology. United States: McGraw Hill; 2004.p.313-8. Chandran S, Sridhar N, Veeranjaneyulu A. Nitric oxide: concepts, current perspectives and future therapeutic implications. Indian J Pharmacol 1998;30:35166. Lee J, Ryu H, Ferrante RJ, Morris SM Jr, Ratan RR. Translational control of inducible nitric oxide synthase expression by arginine can explain the arginine paradox. Proc Natl Acad Sci U S A 2003;100:4843-8. Nakaki T, Hishikawa K.The arginine paradox [Article in Japanese]. Nippon Yakurigaku Zasshi 2002;119:7-14. Alderton WK, Cooper CH, Knowles RG. Nitric oxide synthases: structure, function and inhibition. Biochem J 2001;357:593-615. Khan MA, Thompson CS, Jeremy JY, Mumtaz FH, Mikhailidis P, Morgan RJ. The effect of superoxide dismutase on nitric oxide-mediated and electrical field-stimulated diabetic rabbit cavernosal smooth muscle relaxation. BJU Int 2001;87:98-103. Ichihara A, Hayashi M, Hirota N, Saruta T. Superoxide inhibits neuronal nitric oxide synthase influences on afferent arterioles in spontaneously hypertensive rats. Hypertension 2001;37:630-4. Shoba T, Tay SS. Nitrergic and peptidergic innervation in the developing rat heart. Anat Embryol 2000;201:491-500. Vincent SR. Histochemistry of nitric oxide synthase in the peripheral nervous system. In: Toda N, Moncada S, Furchgott R, Higgs EA, editors. Nitric oxide and the Peripheral Nervous System. London: Portland Press; 2000. Mbaku EN, Zhang L,Duckles SP,Buchholz J. Nitric oxide synthase containing nerves facilitate adrenergic transmitter release in sheep middle cerebral arteries. J Pharmacol Exp Ther 2000;293:397-402. Si ML,Lee TJF. Presynaptic α7-nicotinic acetylcholine receptors mediate nicotine-induced nitric oxidergic neurogenic vasodilatation in porcine basilar arteries. J Pharmacol Exp Ther 2001;298:122-8. Toda N,Ayajiki K, Tanaka T, Okamura T. Preganglionic and postganglionic neurons responsible for cerebral vasodilation mediated by NO in anesthetized dogs. J Cereb Blood Flow Metab 2000;20:700-8. Toda N, Tanaka T, Ayajiki K, Okamura T. Cerebral vasodilatation induced by stimulation of the pterygopalatine ganglion and greater petrosal nerve in anesthetized monkeys. Neuroscience 2000b;96:393-8. Edvinsson L, Elsas T, Suzuki N, Shimizu T, Lee TJ. Origin and colocalization of NOS, CGRP, PACAP, and VIP in the cerebral circulation of the rat. Micros Res Tech 2001;53:221-8. Lucas KA,Pitari GM,Kazerounian S, Ruiz-Stewart I, Park J, Schulz S et al. Guanylyl cyclases and signaling by cyclic GMP. Pharmacol Rev 2000;52:375-
414. 21. De Man JG, De Winter BY, Boeckxstaens GE, Herman AG, Pelckmans PA. Effect of Cu2+ on relaxations to the nitrergic neurotransmitter, NO and Snitrosothiols in the rat gastric fundus. Br J Pharmacol 1996;119:990-6. 22. Mancuso C, Bonsignore A, Di Stasio E, Mordente A, Motterlini R. Bilirubin and S-nitrosothiols interaction: Evidence for a possible role of bilirubin as a scavenger of nitric oxide. Biochem Pharmacol 2003;66:2355-63. 23. Wagner W,Nootbaar-Wagner U. Prophylactic treatment of migraine with gamma-linolenic and alpha- linolenic acids. Cephalalgia 1997;17:127-30. 24. Thomsen LL, Olesen J. Nitric oxide in primary headaches. Curr Opin Neurol 2001;14:315-21. 25. Tepper SJ, Rapoport A, Sheftell F. The pathophysiology of migraine. Neurologist 2001;7:279-86. 26. Ayajiki K, Tanaka T, Okamura T, Toda N. Evidence for nitroxidergic innervation in monkey ophthalmic arteries in vivo and in vitro. Am J Physiol Heart Circ Physiol 2000;279:2006-12. 27. Mizuno K,Koide T,Yoshimura M, Araie M. Neuroprotective effect & intraocular penetration of nipradilol, a beta-blocker with NO donative action. Investig Ophthalmol Vis Sci 2001;42:688-94. 28. Herring N,Golding S, Paterson DJ. Presynaptic NO-cGMP pathway modulated vagal control of heart rate in isolated adult guinea pig atria. J Mol Cell Cardiol 2000;32:1795-804. 29. Choate JK, Danson EJ, Morris JF, Paterson DJ. Peripheral vagal control of heart rate is impaired in nNOS knockout mice. Am J Physiol Heart Circ Physiol 2001;281:2310-7. 30. Andersson KE, Alm P. Neurogenic Nitric oxide and the lower urinary tract. In: Toda N, Moncada S, Furchgott RF, Higgs EA, editors. Nitric oxide and the peripheral nervous system. London: Portland Press; 2000. 31. Shaamash AH, Elsonosy ED, Zakhari MM, Radwan SH, El-Dien HM. Placental NOS activity and NO production in normal pregnancy, pre-eclampsia and eclampsia. Int J Gynaecol Obstet 2001;72:127-33. 32. Steers WD. Neural pathways and central sites involved in penile erection: Neuro-anatomy and clinical implications. Neurosci Biobehav Rev 2000;24:50716. 33. Kirkeby OJ, Kutzsche S, Risoe C, Rise IR. Cerebral nitric oxide concentration and microcirculation during hypercapnia, hypoxia and high intracranial pressure in pigs. J Clin Neurosci 2000;7:531-8. 34. Van Mil AH, Spilt A, Van Buchem MA, Bollen EL, Teppema L, Westendorp RG, et al. Nitric oxide mediates hypoxia-induced cerebral vasodilation in humans. J Appl Physiol 2002;92:962-6. 35. Tanaka T, Ayajiki K, Fujioka H, Toda N, Okamura T. Protection by hypothermia of hypoxia-induced inhibition of neurogenic vasodilation in porcine cerebral arteries. J Pharmacol Sci 2003;92:93-9. 36. White RP, Vallance P, Markus HS. Effect of inhibition of NOS on dynamic cerebral autoregulation in humans. Clin Sci 2000;99:555-60. 37. Wilkens H, Guth A, Konig J, Forestier N, Cremers B, Hennen B, et al. Effect of inhaled iloprost plus oral sildenafil in patients with primary pulmonary hypertension. Circulation 2001;104:1218-22. 38. Santizo R, Baughman VL, Pelligrino DL. Relative contributions from neuronal and endothelial NOS to regional cerebral blood flow changes during forebrain ischaemia in rats. Neuroreport 2000;11:1549-53.
Join “IndPharm” IJP uses “IndPharm” to broadcast announcements. Want to join? Please E-mail:
[email protected] 76
Indian J Pharmacol
| April 2005 | Vol 37 | Issue 2 | 69-76
