Effects of phosphodiesterase inhibitors on oesophageal neuromuscular functions

The cyclic nucleotides adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP) mediate the inhibitory effects of vasoactive intestinal polypeptide and nitric oxide on oesophageal smooth muscle. Phosphodiesterases (PDE) terminate their actions. We hypothesized that PDE inhibitors alter nerve-induced responses of oesophageal and lower oesophageal sphincter (LES) smooth muscle. An electrical field known to activate intrinsic oesophageal nerves was used to stimulate transverse muscle strips from the opossum oesophagus. This produced a contractile off-response from circular oesophageal muscle and a biphasic relaxation of the LES - an initial rapid relaxation (R1) and a slower sustained relaxation (R2). The effects on LES and oesophageal muscle of zaprinast (type V), zardaverine (type III/IV) and theophylline (non-specific) PDE inhibitors were explored. All three PDE inhibitors decreased LES tone and attenuated the off-response. Zaprinast and theophylline increased the latency of the off-response. Zaprinast prolonged R1, and slowed its recovery. It also increased the percentage relaxation of the second R2. Zardaverine increased the percentage relaxation of R2. Theophylline slowed the recovery of R2. PDEs play a role in maintaining LES tone and its recovery after LES relaxation. They may also modulate oesophageal motor activity.     

Possible role of cAMP,cGMP and [Ca2+]i during NANC relaxation in the cat airway smooth muscle

To investigate the possible role of cyclic nucleotides and [Ca²⁺]_i in non-adrenergic non-cholinergic (NANC) relaxations evoked by electrical field stimulation (EFS) in the cat airway, we studied the effects of specific phosphodiesterase (PDE) type IV or V inhibitors on the biphasic-NANC relaxations, the correlation between NANC relaxation and changes in intracellular levels of cAMP and cGMP ([cAMP]_i and [cGMP]_i), and measured changes in [Ca²⁺]_i during the NANC relaxation. EFS (repetitive stimuli at 20 Hz, 1 or 4 ms pulse duration, 50 V) applied to the bronchial smooth muscle during contraction induced by 5-HT (10⁻⁵ M) in the presence of atropine (10⁻⁶ M) and guanethidine (10⁻⁶ M) elicited biphasic NANC relaxations. Zaprinast (>3×10⁻⁷ M), a specific PDE type V inhibitor, preferentially enhanced the amplitude of the first component of the NANC relaxations. However, rolipram (>3×10⁻⁷ M) enhanced both the first and second component of the NANC relaxation to a similar extent. In the trachea, EFS evoked monophasic NANC relaxation accompanied by a concomitant accumulation of [cAMP]_i and [cGMP]_i. Pretreatment with rolipram (3×10⁻⁶ M) enhanced the accumulation of [cAMP]_i and amplitude of NANC relaxation evoked by EFS. However, zaprinast did not affect the amplitude of NANC relaxation although it significantly increased the levels of [cGMP]_i. N^ω-Nitro-l-arginine methylester (l-NAME; 10⁻⁵ M) completely suppressed the accumulation of [cGMP]_i but only partly suppressed the NANC relaxation evoked by EFS. In contrast, EFS significantly enhanced [cAMP]_i in the presence of l-NAME. During NANC relaxation, time-dependent decrease in [Ca²⁺]_i occurred, which was partly suppressed by l-NAME. These results indicate that NANC relaxation is associated with concomitant accumulation in both [cAMP]_i and [cGMP]_i, and decrease in [Ca²⁺]_i. However, the timing of the action of [cGMP]_i and [cAMP]_i in NANC relaxations differs in the central and peripheral airway.     

Cyclic Nucleotides Increase During Neuronally Induced Relaxation of Sphincteric and Nonsphincteric Gastrointestinal Smooth Muscle

Neuronal stimulation of most isolated precontracted gastrointestinal smooth muscle results in relaxation. This study examined the changes in cyclic nucleotide content associated with neuronally induced relaxation in sphincteric and nonsphincteric smooth muscle of the gut. Electrical field stimulation produced a frequency-dependent relaxation of the guinea pig proximal colon (EF₅₀= 1.6 Hz) and taenia coli (EF₅₀= 2.4 HZ), and the canine proximal colon (EF₅₀= 3.1 Hz) and internal anal sphincter (EF₅₀= 3.0 Hz). Changes in cyclic nucleotide content occurred during EFS-induced relaxation. Cyclic AMP levels were significantly increased in the guinea pig poximal colon and taenia coli and in the canine proximal colon. No change in cyclic GMP occurred in these smooth muscles. Conversely, in the canine internal anal sphincter the cyclic GMP content increased significantly with no change in cyclic AMP. These effects in this sphincter are similar to those identified previously in the lower esophageal sphincter of opossum, dog, and man. Tetrodotoxin blocked the EFS-induced relaxation and the associated accumulation of cyclic nucleotides of all tissues. Therefore, EFS-induced relaxation is mediated by the activation of neurons and the release of inhibitory neurotransmitter(s). Also, EFS-induced relaxation is closely associated with increases in smooth muscle cyclic nucleotide content. Most importantly, the change in cyclic nucleotide accumulation that occurs during EFS-induced relaxation is different for sphincteric and nonsphincteric smooth muscle, (i.e., different cyclic nucleotides are generated in sphincteric and nonsphincteric smooth muscle in response to stimulation of nonadrenergic, nonchohnergic, inhibitory enteric neurons).     

Endothelin A receptors mediate relaxation of guinea pig internal anal sphincter through cGMP pathway

Background Endothelin (ET) modulates motility of the internal anal sphincter through unclear receptor subtypes. Methods We measured relaxation of guinea pig internal anal sphincter strips caused by ET‐related peptides and binding of ¹²⁵I‐ET‐1 to cell membranes prepared from the internal anal sphincter muscle. Visualization of ¹²⁵I‐ET‐1 binding sites in tissue was performed by autoradiography. Key Results In the guinea pig internal anal sphincter, ET‐1 caused a marked relaxation insensitive to tetrodotoxin, atropine, or ω‐conotoxin GVIA. ET‐2 was as potent as ET‐1. ET‐3 caused a mild relaxation. The relative potencies for ETs to cause relaxation were ET‐1 = ET‐2 > ET‐3. The ET‐1‐induced relaxation was inhibited by BQ‐123, an ET_A antagonist, but not by BQ‐788, an ET_B antagonist. These indicate that ET_A receptors mediate the relaxation. The relaxant response of ET‐1 was attenuated by LY 83583, KT 5823, Rp‐8CPT‐cGMPS, tetraethyl ammonium, 4‐aminopyridine and N(omega)‐nitro‐l‐arginine, but not significantly affected by N^G‐nitro‐l‐arginine methyl ester, N^G‐methyl‐l‐arginine, charybdotoxin, apamin, KT 5720, and Rp‐cAMPS. These suggest the involvement of cyclic guanosine 3′,5′‐cyclic monophosphate (cGMP), and potassium channels. Autoradiography localized ¹²⁵I‐ET‐1 binding to the internal anal sphincter. Binding of ¹²⁵I‐ET‐1 to the cell membranes prepared from the internal anal sphincter revealed the presence of two subtypes of ET receptors, ET_A and ET_B receptors. Conclusions & Inferences Taken together, these results demonstrate that ET_A receptors mediate relaxation of guinea pig internal anal sphincter through the cGMP pathway.     

Investigation of the role of adrenergic and non-nitrergic, non-adrenergic neurotransmission in the sheep isolated internal anal sphincter

Abstract Nitric oxide is widely established as an important neurotransmitter in the control of anal sphincter tone; although, a number of other transmitters have also been tentatively implicated. Whilst α‐adrenoceptor antagonists reduce anal sphincter pressure in man, the role of noradrenaline as a possible transmitter is poorly characterised. We have investigated the contribution of these transmitters to neurogenic relaxations, and evaluated the possible role of a non‐nitrergic, non‐adrenergic transmitter. The magnitude and duration of neurogenic responses were examined by measuring responses to electrical field stimulation (EFS) in segments of sheep internal anal sphincter following the development of spontaneous myogenic tone. Neurogenic relaxations induced by EFS were significantly reduced in the presence of N^G‐nitro‐L‐arginine methyl ester (l ‐NAME) suggesting major involvement of nitric oxide as a neurotransmitter. The duration of neurogenic relaxations was inversely related to the frequency of EFS, with contractile responses often manifest at higher frequencies. The duration of relaxations at high frequencies of EFS was increased by bretylium (adrenergic neurone blocker) and prazosin (α₁‐adrenoceptor antagonist). At higher frequencies of EFS, 60% of preparations also produced a residual non‐nitrergic, non‐adrenergic, apamin‐sensitive relaxation which was unaffected by vasoactive intestinal polypeptide (VIP) and inhibitors of purinergic responses [suramin, pyridoxal‐phosphate‐6‐azophenyl 2′,4′ disulfonic acid (PPADS) and α,β‐methylene adenosine triphosphate (ATP)]. However, MRS2179 (P2Y₁ receptor antagonist) showed a modest inhibitory effect. We conclude that endogenous noradrenaline acts via postjunctional α₁‐adrenoceptors to antagonize neurogenic relaxations that are largely mediated by nitric oxide. Our results indicate the involvement of a non‐nitrergic, non‐adrenergic, apamin‐sensitive transmitter which is inhibited by MRS2179, suggesting a possible role for purines.     

Involvement of cyclic guanosine monophosphosphate (cGMP) and cytosolic guanylate cyclase in the regulation of synaptic ribbon numbers in rat pineal gland

In the rat pineal gland N-acetyltransferase (NAT) activity and synaptic ribbon (SR) numbers display a circadian rhythm. It is well-known that NAT activity is regulated by adrenergic mechanisms involving cyclic adenosine monophosphate (cAMP) as a second messenger. However, the mechanism involved in the regulation of SR numbers has not been established so far. In the present in vitro study, we have investigated the effects of 8-bromo-cyclic guanosine monophosphate (8-bromo-cGMP), a cyclic guanosine monophosphate (cGMP) analog, and stimulation of guanylate cyclase on SR numbers. Incubation with 8-bromo-cGMP increased SR numbers in a dose- and time-dependent manner. Further, stimulation of the cytosolic guanylate cyclase also resulted in increased SR numbers. Adrenergic agonists stimulated cGMP but did not alter SR numbers. These findings suggest that cGMP is involved as a second messenger in the regulation of SR numbers. Since the adrenergically stimulated increase in cGMP did not influence SR numbers, a non-adrenergic cGMP metabolic pathway seems to be involved in the regulation of SR numbers in the rat pineal gland.     

外源性NO与内源性NO对海马区cGMP合成的影响

正常沙土鼠海马脑片孵化时加用硝普钠及钳夹沙土鼠的双侧颈总动脉制造脑缺血模型.硝普钠增加cGMP的产生,cGMP主要分布在海马本部曲张的纤维及少数的细胞体中.脑缺血再灌流后增加cGMP的产生,cGMP主要分布在海马本部的细胞体及少量纤维中.认为外源性NO与内源性NO介导海马区cGMP的合成分布不同.     

Species dependent differences in the actions of sympathetic nerves and noradrenaline in the internal anal sphincter

Excitatory motor innervation to the internal anal sphincter (IAS) of the monkey, the rabbit and mouse were compared. Contractile responses to electrical field stimulation of nerves (EFS, atropine 1 micromol L(-1) and N(omega)-nitro-L-arginine 100 micromol L(-1) present throughout) were examined in isolated strips of IAS. In the monkey IAS, EFS caused frequency dependent (1-30 Hz) contractions which were abolished by guanethidine (10 micromol L(-1)) or phentolamine (3 micromol L(-1)). The sympathetic neurotransmitter noradrenaline (NA) also caused concentration-dependent (10 nmol L(-1)-100 micromol L(-1)) contractions which were abolished by phentolamine revealing a small relaxation that was abolished by propranolol (3 micromol L(-1)). In contrast, EFS caused only relaxation of the mouse and rabbit IAS which was not affected by guanethidine. Furthermore, NA relaxed these muscles and relaxation was nearly abolished by combined addition of phentolamine and propranolol. In conclusion, the monkey IAS is functionally innervated by sympathetic nerves that contract the muscle via excitatory alpha-adrenergic receptors. In contrast, no significant motor function could be identified for sympathetic nerves in the rabbit or mouse IAS although adrenergic receptors linked to muscle inhibition are present. These data reveal species dependent differences in sympathetic motor innervation and suggest that some species are more appropriate than others as models for motor innervation to the human IAS.     

Nitrergic–purinergic interactions in rat distal colon motility

Responses of rat distal colon circular muscle strips to exogenous nitric oxide (NO) and adenosine 5'-triphosphate (ATP) and to electrical field stimulation (EFS) were assessed in the absence/presence of various agents that interfere with nitrergic-purinergic pathways. Exogenous NO (10-6 to 10-4 mol L-1) elicited concentration-dependent, tetrodotoxin (TTX)-insensitive relaxations. The soluble guanylyl-cyclase (sGC) inhibitor 1H[1,2,4,]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) reduced duration and amplitude; the small conductance Ca2+-sensitive K+ (SK)-channel blocker apamin (APA) only shortened the relaxations. ODQ + APA showed a marked inhibitory effect on duration and amplitude. TTX, APA, the NO-synthase inhibitor N(omega)-nitro-l-arginine methyl ester (l-NAME) and the purinergic receptor P2Y antagonist Reactive Blue 2 (RB2) shortened the relaxations by exogenous ATP (10-3 mol L-1) but did not influence the amplitude. ODQ had no effect. TTX + l-NAME did not yield a more pronounced inhibitory effect than TTX alone. The effect of ATP-gamma-S was similar to that of ATP. Electrical field stimulation (EFS) (40 V, 0.05 ms, 0.5-4 Hz for 30 s) yielded TTX-sensitive relaxations that were not altered by l-NAME, ODQ or RB2. APA shortened the relaxations. l-NAME + APA nearly abolished these relaxations. ODQ + APA and RB2 +l-NAME reduced the duration. These results suggest that distinct sets of small conductance SK-channels are involved in the amplitude and the duration of the relaxations and that NO increases their sensitivity to NO and ATP via guanosine 3',5'-cyclic monophosphate (cGMP). ATP elicits relaxations via P2Y receptors with subsequent activation of SK-channels and induces neuronal release of NO. Both nitrergic and purinergic pathways must be blocked to inhibit EFS-induced relaxations.     

Studies on inhibition of human platelet function by sodium nitroprusside. Kinetic evaluation of the effect on aggregation and cyclic nucleotide content

In this study, we explored the ability of sodium nitroprusside to inhibit the aggregation of human platelets in platelet-rich plasma (PRP) and whole blood and its effects on intracellular levels of guanosine 3',5'-cyclic monophosphate (cGMP) and adenosine 3',5'-cyclic monophosphate (cAMP). The experiments investigated dose-dependent effects of nitroprusside starting from concentrations in the range of circulating levels achievable in vivo during drug administration in humans. Furthermore, we investigated the time-course of both antiaggregating action and the influence on cyclic nucleotide synthesis. Results showed that sodium nitroprusside inhibited the aggregation induced by adenosine 5-diphosphate (ADP) and collagen starting from concentration as low as 2 micromol/l. The IC(50) value for ADP-induced aggregation in PRP was 18.7+/-2.4 micromol/l. The inhibition of platelet aggregation showed a time-dependent behaviour and was not reversible within 90 min. The accumulation of intraplatelet cGMP in the presence of sodium nitroprusside exhibited a comparable time-course characterized by an early increase, a steady state and a late further increase. The time-course of cAMP synthesis was very similar to that of cGMP. Our data evidenced a long-lasting inhibition of platelet responses by sodium nitroprusside and excluded a desensitization of platelet guanylyl cyclase after 3-h exposure to nitric oxide (NO). Furthermore, they indicated a role of cAMP accumulation in the antiaggregating effects of nitroso donor: the simultaneous increase of intracellular content of cAMP and cGMP can synergize in the reduction of the platelet responses.     

Inactivation of platelet PDE2 by an affinity label: 8-[(4-bromo-2, 3-dioxobutyl)thio]cAMP

Cyclic GMP-stimulated cyclic nucleotide phosphodiesterase (PDE2) is the second most abundant of this class of enzymes in platelets. PDE2 probably plays an important role in the regulation of elevated intracellular concentrations of cAMP and cGMP in platelets inhibited by prostacyclin and/or nitric oxide. The cAMP and cGMP PDEs have catalytic domains with 28-40% identity, but vary in their substrate specificity and affinity. As a first step toward the goal of identifying important amino acids in the substrate binding site pocket, we have employed the affinity analog 8-[(4-bromo-2, 3-dioxobutyl)thio]adenosine-3'5' cyclic monophosphate (8-BDB-TcAMP) to inactivate PDE2 and observe the pattern of protection by substrates and their products. Incubation of purified platelet PDE2 with 8-BDB-TcAMP (2-10 mM) resulted in a time-dependent, irreversible inactivation of the enzyme with a second-order rate constant of 0.013 min(-1) mM(-1). Both substrates, cAMP and cGMP, as well as the products of hydrolysis by PDE2, AMP and GMP, exhibited concentration-dependent protection against inhibition by 8-BDB-TcAMP, but no protection was noted with ADP or ATP, which are not hydrolyzed by the enzyme. This compound, 8-BDB-TcAMP, and similar affinity reagents should prove useful in delineating amino acids in the active site of PDE2.     

5-HT4 receptor agonists enhance both cholinergic and nitrergic activities in human isolated colon circular muscle

Previous studies have demonstrated mixed inhibitory and facilitatory effects of 5-hydroxytryptamine-4 (5-HT(4)) receptor agonists on electrical field stimulation (EFS)-induced responses in human isolated colon. Here we report three types of responses to EFS in human isolated colon circular muscle: monophasic cholinergic contraction during EFS, biphasic response (nitrergic relaxation during EFS followed by cholinergic contraction after termination of EFS) and triphasic response (cholinergic contraction followed by nitrergic relaxation during EFS and a tachykininergic contraction after EFS). The effects of two 5-HT(4) receptor agonists, prucalopride and tegaserod were then investigated on monophasic responses only. Each compound inhibited contractions during EFS in a concentration-dependent manner. In the presence of N(omega)-nitro-l-arginine methyl ester (l-NAME) however, prucalopride and tegaserod enhanced the contractions in a concentration-dependent manner. In strips where the tone was elevated with substance-P and treated with scopolamine, EFS-induced relaxations were enhanced by the two agonists. The above observed effects by the two agonists were abolished by 5-HT(4) receptor antagonist SB-204070. The two agonists did not alter the tone raised by substance-P in the presence of scopolamine and l-NAME and did not affect carbachol-induced contractions in the presence of tetrodotoxin. These results suggest that in the circular muscle of human colon, 5-HT(4) receptor agonists simultaneously facilitate the activity of neurones which release the inhibitory and excitatory neurotransmitters, nitric oxide and acetylcholine respectively.     

Cyclic 3'',5''-adenosine monophosphate mimics slow synaptic excitation in myenteric plexus neurons

Iontophoretic injection of cyclic adenosine monophosphate (cAMP) into the somas of ganglion cells in the myenteric plexus of guinea pig small intestine mimicked the slow excitatory postsynaptic potentials that occur in these neurons. These effects were: (1) membrane depolarization. (2) decreased membrane conductance, (3) augmented excitability with increased probability of spontaneous spike discharge and increased spike discharge during depolarizing current pulses, (4) anodal break excitation, (5) suppression of hyperpolarizing afterpotentials. The same effects were produced by application of membrane-permeable analogs of cAMP in the bathing medium. The phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine, potentiated the effects of the analogs. The results suggest that the biochemical basis for long-lasting excitatory neuromodulation in myenteric neurons is activation of adenylate cyclase and elevation of intraneuronal cAMP.     

Hemolysate inhibits cerebral artery relaxation

In helical strips of dog middle cerebral arteries partially contracted with prostaglandin (PG) F2 alpha, relaxations induced by angiotensin-II, possibly mediated by PGI2, and those induced by PGH2 were reversed to a contraction or markedly reduced by treatment with hemolysate, which, however, attenuated the PGI2-induced relaxation only slightly. The relaxant response of human middle cerebral arterial strips to PGH2 was also suppressed by hemolysate. Dog and monkey middle cerebral arteries responded to transmural electrical stimulation and nicotine with transient relaxations, which were quite susceptible to tetrodotoxin and hexamethonium, respectively; the relaxations were abolished almost completely by hemolysate and methylene blue. On the other hand, the relaxant response of dog cerebral arteries to a low concentration of K+ was not influenced by hemolysate or by methylene blue, but was reversed to a contraction by treatment with ouabain. Relaxations induced by substance-P and nitroglycerin were markedly inhibited by hemolysate; removal of endothelium abolished the relaxation by substance-P, but did not influence the nitroglycerin-induced relaxation. Hemolysate may interfere with the biosynthesis of PGI2 in the vascular wall, thereby reversing the relaxation induced by angiotensin-II and PGH2 to a contraction. Relaxations induced by electrical and chemical stimulation of vasodilator nerves innervating cerebral arteries appear to be elicited by a mechanism dependent on cellular cyclic guanosine monophosphate (GMP), like that underlying the substance-P-induced and nitroglycerin-induced relaxation. These actions of hemolysate may be involved in the genesis of cerebral vasospasm after subarachnoid hemorrhage.     

Ambulatory high-resolution manometry, lower esophageal sphincter lift and transient lower esophageal sphincter relaxation

Background Lower esophageal sphincter (LES) lift seen on high‐resolution manometry (HRM) is a possible surrogate marker of the longitudinal muscle contraction of the esophagus. Recent studies suggest that longitudinal muscle contraction of the esophagus induces LES relaxation. Aim Our goal was to determine: (i) the feasibility of prolonged ambulatory HRM and (ii) to detect LES lift with LES relaxation using ambulatory HRM color isobaric contour plots. Methods In vitro validation studies were performed to determine the accuracy of HRM technique in detecting axial movement of the LES. Eight healthy normal volunteers were studied using a custom designed HRM catheter and a 16 channel data recorder, in the ambulatory setting of subject’s home environment. Color HRM plots were analyzed to determine the LES lift during swallow‐induced LES relaxation as well as during complete and incomplete transient LES relaxations (TLESR). Key Results Satisfactory recordings were obtained for 16 h in all subjects. LES lift was small (2 mm) in association with swallow‐induced LES relaxation. LES lift could not be measured during complete TLESR as the LES is not identified on the HRM color isobaric contour plot once it is fully relaxed. On the other hand, LES lift, mean 8.4 ± 0.6 mm, range: 4–18 mm was seen with incomplete TLESRs (n = 80). Conclusions & Inferences Our study demonstrates the feasibility of prolonged ambulatory HRM recordings. Similar to a complete TLESR, longitudinal muscle contraction of the distal esophagus occurs during incomplete TLESRs, which can be detected by the HRM. Using prolonged ambulatory HRM, future studies may investigate the temporal correlation between abnormal longitudinal muscle contraction and esophageal symptoms.     

Regulation of adipokinetic hormone release from locust neuroendocrine tissue: participation of calcium and cyclic AMP

In the locust, cyclic adenosine monophosphate (cAMP) mediates at least part of the effects of octopamine, the neurotransmitter which regulates the release of two adipokinetic hormones (AKHs) from the glandular lobe of the corpus cardiacum (CC). We have examined the requirement for extracellular Ca2+ in the process of AKH release mediated by octopamine and by agents which artificially elevate intracellular cAMP levels. Octopamine and the adenylate cyclase activator forskolin elevate the cAMP content of the glandular lobe in normal saline, in normal saline with the Ca2+ channel blocker, methoxyverapamil, and in Ca2+-free saline during 10-min exposure periods. Octopamine, forskolin, and 8-bromo cAMP mediate release of AKHs in vitro in normal saline, but release is prevented in the absence of extracellular Ca2+. When glands are exposed to these agents in normal saline in the presence of methoxyverapamil, AKH release is curtailed in a similar manner. Lanthanum and EGTA dramatically reduce cAMP production elicited by octopamine and forskolin, and lanthanum prevents octopamine-mediated release of AKHs. The phosphodiesterase inhibitor, IBMX, elevates cAMP content in the presence and absence of extracellular Ca2+, and stimulates normal release of AKHs both in the presence and absence of extracellular Ca2+. However, following extensive washing in Ca2+-free saline, IBMX fails to evoke AKH release. Methoxyverapamil has no effect on IBMX-mediated secretion. These results suggest that IBMX may mobilize intracellular stores of Ca2+ to induce release. Extracellular Ca2+ is apparently required for the process of neurotransmitter-evoked release, as has been shown for release of other peptide hormones. Cyclic AMP is intimately associated with Ca2+ in mediating this process. The release of AKHs is more dependent upon extracellular Ca2+ than is cAMP production under the conditions examined in this study. Ca2+ may provide the signal which initiates the secretory response, although cAMP may modulate this signal or the cells' responsiveness to this signal in some way. Support for this hypothesis is provided by experiments with the Ca2+ ionophore, A23187. This agent provokes release of AKHs in a Ca2+-dependent manner, probably by elevating intracellular Ca2+ levels. A23187 does not elevate cAMP levels in the glandular lobe, indicating that cAMP elevation is not a prerequisite for secretion.     

CSF cyclic nucleotides and somatostatin in Parkinson's disease

Concentrations of cyclic adenosine 3',5' monophosphate (cAMP) were significantly lower in parkinsonian patients than in controls, but concentrations of guanosine 3',5' monophosphate (cGMP) were not altered. Both cAMP and cGMP levels were lower in patients with more severe symptoms on the left side of the body. Somatostatin-like immunoreactivity (SLI) was similar in parkinsonian patients and controls. Both cAMP and SLI were significantly related to acetylcholinesterase activity.     

Lowered cAMP and cGMP signalling in the brain during levodopa-induced dyskinesias in hemiparkinsonian rats: new aspects in the pathogenetic mechanisms

Dysregulation of dopamine receptors is thought to underlie levodopa-induced dyskinesias in experimental models of Parkinson's disease. It is unknown whether an imbalance of the second messengers, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), is involved in the alterations of levodopa/dopamine signal transduction. We examined cAMP and cGMP signalling in the interconnected cortico-striatal-pallidal loop at the peak of levodopa-induced dyskinesias in rats with 6-hydroxydopamine lesions in the substantia nigra. In addition, we examined the role of phosphodiesterase (PDE) and the rate of cAMP and cGMP degradation on the severity of levodopa-induced dyskinesias in animals pretreated with PDE inhibitor, zaprinast. Unilateral lesion of substantia nigra led to an increase in cAMP but a decrease in cGMP levels in the ipsilateral basal ganglia. After chronic levodopa treatment, cAMP and cGMP were differentially regulated in eukinetic animals: the cAMP level increased in the cortex and striatum but decreased in the globus pallidus of both hemispheres, whereas the cGMP decreased below baseline levels in the contralateral cortico-striatal-pallidal regions. In dyskinetic animals chronic levodopa treatment led to an absolute decrease in cAMP and cGMP levels in cortico-striatal-pallidal regions of both hemispheres. Pretreatment with zaprinast reduced the severity of levodopa-induced dyskinesias, and partly prevented the decrease in cyclic nucleotides compared with pretreatment with saline-levodopa. In conclusion, using a rat model of hemiparkinsonism, we observed a significant reduction in the levels of cyclic nucleotides in both hemispheres at the peak of levodopa-induced dyskinesias. We propose that such a decrease in cyclic nucleotides may partly result from increased catabolism through PDE overactivity.     

Comparison of spontaneously released endothelium-derived relaxing factor in cerebral and extracerebral arteries in rabbits

Abstract

To investigate regional differences in spontaneously released endothelium-derived relaxing factor (EDRF), a bioassay of spontaneously released EDRF was performed on rabbit basilar, ear; common carotid and thoracic arteries using an isometric tension measurement technique and a measurement of cyclic guanosine monophosphate (cCMP) content in the vascular smooth muscle. The amount of spontaneously released EDRF was higher in the basilar artery than in any other arteries examined (p < 0.01). The levels of cCMP were 57.3 ± 4.4 (n = 7) in basilar, 26.5±4.3 (n = 6) in ear; 24.5±2.3 (n = 11) in common carotidand 30.3 ±3.8 pmol/g tissue (n = 8) in thoracic artery with endothelium, while endothelium-denuded arteries showed 24.2 ± 6.6 (n = 5), 17.5 ± 5.1 (n = 6), 20.1 ± 2.9 (n = 7) and 14.4 ± 2.3 pmol/g tissue (n = 8) in the same order. Haemoglobin (10~5 m, incubated with the artery for 5 min, significantly reduced the level of cGMP in all vessels with endothelium: 35.3±4.4 (basilar), 76.0 + 2.7 (ear), 14.0±1.9 (common carotid) and 8.7 ±1.2 pmol/g tissue (thoracic artery). Since endothelium-dependent relaxation is associated with a rise in the cGMP content of the smooth muscle cell, the data of cGMP measurement in addition to the bioassay of spontaneously released EDRF in tension measurement suggests that the spontaneous release of EDRF is much greater in the basilar artery than in extracerebral arteries. It is concluded that the intensity of the spontaneously released EDRF is relatively higher in the intracerebral artery than in the extracerebral artery. [Neurol Res 1993; 15: 327-332]     

Neuropeptide effects on rat chondrocytes and perichondrial cells in vitro

This study examines if cultured chondrocytes and perichondrial cells change the level of cAMP and/or cGMP in response to application of the neuropeptide calcitonin gene-related peptide (CGRP). Cells collected from the knee region of 4-8 days old rat pups were cultured in vitro. Cultures were exposed to 10(-10)-10(-6) M CGRP during 10 minutes. The levels of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) in the cultures and in controls were determined by radioimmunoassay. The results show that application of CGRP causes a distinctly increased level of cAMP, that was absent when CGRP was applied together with the CGRP(1) receptor antagonist. The level of cGMP was not obviously altered. Hence, it is possible that terminals of primary sensory neurones present in developing cartilage influence chondrocytes and perichondrial cells via local release of CGRP.