Increase In 3H Efflux Research Articles

Induction by low Na+ or Cl- of cocaine sensitive carrier-mediated efflux of amines from cells transfected with the cloned human catecholamine transporters.

1. COS-7 cells transfected with the cDNA of the human dopamine transporter (DAT cells) or the human noradrenaline transporter (NAT cells) were loaded with [3H]-dopamine or [3H]-noradrenaline and superfused with buffers of different ionic composition. 2. In DAT cells lowering the Na+ concentration to 0, 5 or 10 mM caused an increase in 3H-efflux. Cocaine (10 microM) or mazindol (0.3 microM) blocked the efflux at low Na+, but not at 0 Na+. Lowering the Cl- concentration to 0, 5 or 10 mM resulted in an increased efflux, which was blocked by cocaine or mazindol. Desipramine (0.1 microM) was without effect in all the conditions tested. 3. In NAT cells, lowering the Na+ concentration to 0, 5 or 10 mM caused an increase in 3H-efflux, which was blocked by cocaine or mazindol. Desipramine produced a partial block, its action being stronger at 5 or 10 mM Na+ than at 0 mM Na+. Efflux induced by 0, 5 or 10 mM Cl- was completely blocked by all three uptake inhibitors. 4. In cross-loading experiments, 5 mM Na(+)- or 0 Cl(-)-induced efflux was much lower from [3H]-noradrenaline-loaded DAT, than NAT cells and was sensitive to mazindol, but not to desipramine. Efflux from [3H]-dopamine-loaded NAT cells elicited by 5 mM Na+ or 0 Cl- was blocked by mazindol, as well as by desipramine. 5. Thus cloned catecholamine transporters display carrier-mediated efflux of amines if challenged by lowering the extracellular Na+ or Cl-, whilst retaining their pharmacological profile. The transporters differ with regard to the ion dependence of the blockade of reverse transport by uptake inhibitors.

Novel pharmacological sensitivity of the presynaptic calcium channels controlling acetylcholine release in skate electric organ.

The presynaptic terminals of skate (Raja montagui) electric organ were tested for their sensitivity to calcium channel antagonists. Acetylcholine (ACh) release and the elevation of intraterminal Ca2+ concentrations triggered by K+ depolarisation were studied. ACh release was measured as 3H efflux from slices of organ prelabelled with [3H]choline. Depolarisation caused a marked, Ca(2+)-dependent increase in 3H efflux that was completely blocked by 100 microM Cd2+ and by 300 nM omega-conotoxin-MVIIC (MVIIC). Inhibition by MVIIC was concentration dependent (IC50 of approximately 20 nM) and reversible. No inhibition was seen with nifedipine (5 microM) or the two other peptide antagonists studied: omega-conotoxin-GVIA (GVIA) at 5 microM and omega-agatoxin-IVA (Aga-IVA) at 1 microM. In a "nerve plate" preparation (a presynaptic plexus of nerve fibres, Schwann cells, and nerve terminals) changes in intraterminal Ca2+ concentrations were measured by microfluorimetry using fluo-3. An increase in fluorescence, indicating a rise in the free [Ca2+], rapidly followed K+ depolarisation, and this change was restricted to the nerve terminals. This response was insensitive to nifedipine (5 microM), GVIA (5 microM), and Aga-IVA (300 nM) but almost completely abolished by MVIIC (1 microM). MVIIC inhibition was concentration dependent and partially reversible. These results show that the nerve terminals in skate electric organ have calcium channels with a pharmacological sensitivity that is markedly different from the established L, N, and P types in other systems but shares some, but not all, of the features of the recently described Q type.

Comparison between sympathetic adrenergic and purinergic transmission in the dog mesenteric artery.

1. Electrical transmural stimulation evoked a sympathetic contraction in the isolated dog mesenteric artery. This contraction consisted of adrenergic and purinergic components, which were separately observed under conditions where postjunctional P2 purinoceptors were desensitized with alpha, beta-methylene ATP or postjunctional alpha 1 adrenoceptors were blocked by prazosin, respectively. 2. The purinergic component was transient and developed immediately after the start of stimulation and declined rapidly after a peak. In contrast, the adrenergic contraction slowly developed and lasted longer than the purinergic component. Thus, the purinergic and adrenergic components predominantly contributed to the early and later phases of the total sympathetic response, respectively. 3. The peak amplitudes of contraction of both components were similar at 1 Hz, but the adrenergic component occurred more dominantly in the responses to higher frequency stimulation. 4. Cocaine, a neuronal uptake inhibitor of noradrenaline, potentiated the adrenergic component but attenuated the purinergic component. This attenuation was reversed by DG-5128, a prejunctional alpha 2 adrenoceptor antagonist. Treatment with DG-5128 alone augmented both adrenergic and purinergic components. 5. 8-Phenyltheophylline, a P1 purinoceptor antagonist, potentiated the purinergic component without affecting the adrenergic component. 6. Exogenous noradrenaline produced a sustained contraction, which was potentiated by cocaine and was competitively inhibited by prazosin. alpha, beta-Methylene ATP and 8-phenyltheophylline had no effect on the response to noradrenaline. Exogenous ATP produced a transient contraction, which was abolished under conditions where postjunctional P2 purinoceptors were desensitized with alpha, beta-methylene ATP. 8-Phenyltheophylline potentiated but cocaine or prazosin did not affect the ATP response. 7. Electrical stimulation produced an increase in 3H efflux from the sympathetic nerve terminals in the mesenteric arteries pre-incubated with [3H]noradrenaline. The evoked efflux was significantly augmented by cocaine or DG-5128 and was inhibited by guanethidine or tetrodotoxin. alpha, beta-Methylene ATP and 8-phenyltheophylline were without effect. Adenosine reduced the 3H efflux and the inhibition was suppressed by 8-phenyltheophylline. 8. These results suggest that sympathetic contraction of the dog mesenteric artery is caused through not only adrenergic but also purinergic mechanisms, and that both the sympathetic transmissions are predominantly modulated through prejunctional adrenergic mechanisms.

Stimulatory effects of neuronally released norepinephrine on renin release in vitro.

Extracellular high potassium inhibits renin release in vitro by increasing calcium concentrations in the juxtaglomerular cells. We found that the decreased response of renin release from rat kidney cortical slices in high potassium solution (20-80 mM) changed to a strikingly increased one in the presence of nifedipine at doses over 10(-6) M. We then examined the stimulatory effect of extracellular high potassium in the presence of nifedipine on renin release. The enhancement of release was significantly suppressed either by propranolol or by metoprolol but not by prazosin. High potassium plus nifedipine-induced increase in renin release was markedly attenuated by renal denervation. The enhancing effect was not observed when the slices were incubated in calcium-free medium. Divalent cations such as Cd2+, Co2+, and Mn2+ (0.1-3.0 mM) blocked this enhancement in a concentration-dependent manner. High potassium elicited an increase in 3H efflux from the slices preloaded with [3H]norepinephrine. The increasing effect was not influenced by nifedipine but was abolished by the removal of extracellular calcium or by the addition of divalent cations. These observations suggest to us that the high potassium plus nifedipine-induced increase in renin release from the slices is mediated by norepinephrine derived from renal sympathetic nerves and that this neuronally released norepinephrine stimulates renin release via activation of beta-adrenoceptors.

Phosphoinositide hydrolysis and insulin release from isolated perifused rat islets. Studies with glucose.

The ability of glucose to promote the hydrolysis of prelabeled [2-3H]inositol-containing phosphoinositides (PI) was assessed by measuring the efflux of 3H in response to glucose and the accumulation of labeled inositol phosphates. The inclusion of nonradioactive inositol (1 mM) in the perifusion medium dramatically improved our ability to monitor glucose-induced increases in 3H efflux. Efflux studies with this method revealed the following. 1) 3H efflux is significantly greater at 7 than at 2.75 mM glucose, and this parallels a small but significant increase in insulin secretion. 2) D-manno-Heptulose reduces 3H efflux with 7 mM glucose to a level approximating that seen in the presence of 2.75 mM glucose and has no effect on 3H efflux with 2.75 mM glucose. 3) In the presence of 20 mM glucose plus 1 mM inositol, 3H efflux is rapid and biphasic, a response that parallels the timing and amplitude of the biphasic pattern of insulin secretion. Direct measurements of labeled inositol and inositol phosphate levels in islets revealed the following. 4) After 50 min of perifusion with 2.75 or 7 mM glucose, labeled inositol phosphates were significantly greater with 7 mM glucose. 5) In response to 20 mM glucose alone, islet levels of free inositol, inositol monophosphate (IP1), and inositol bisphosphate (IP2) increased. 6) In response to 20 mM glucose plus 1 mM cold inositol, islet levels of free inositol increased, whereas islet levels of IP1, IP2, and inositol trisphosphate (IP3) were reduced compared with values obtained with 20 mM glucose alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Synergistic impact of cholecystokinin and gastric inhibitory polypeptide on the regulation of insulin secretion

The modulation of insulin output from isolated perifused rat islets by the intestinal peptides cholecystokinin (CCK) and gastric inhibitory polypeptide (GIP) was assessed. In the presence of 7 mmol/L (126 mg/dL) glucose, but not 2.75 mmol/L (50 mg/dL) glucose, CCK (5 nmol/L) or GIP (50 ng/mL) alone evoke small insulin secretory responses. However, the combination of GIP (50 ng/mL) plus CCK (5 nmol/L) together with 7 mmol/L glucose results in a markedly amplified insulin secretory response. CCK (50 nmol/L) alone increases phosphoinositide (PI) hydrolysis in islets, an event reflected by an increase in 3H efflux from myo[2- 3H]inositol prelabeled islets and parallel accumulations of labeled inositol phosphates. GIP (50 ng/mL) alone has no effect on PI hydrolysis. However, GIP reduces the quantitative impact of CCK on PI turnover, an effect attributable to the capacity of GIP to elevate islet cAMP levels. CCK has no significant effect on islet cAMP levels. The results support the concept that the synergistic action of these peptides on insulin output is mediated by their ability to generate separate beta cell second messenger molecules. Nutrient-regulated intestinal release of various peptides represents a remarkable control system to ensure the release of insulin from the beta cell in amounts commensurate with both the quantity and quality of nutrient intake.

Time-dependent potentiation of insulin release induced by alpha-ketoisocaproate and leucine in rats: possible involvement of phosphoinositide hydrolysis.

The ability of the amino acid leucine and its keto acid, alpha-ketoisocaproate, to induce insulin release, to initiate phosphoinositide hydrolysis, and to amplify the subsequent insulin secretory response to glucose was assessed. In islets whose inositol-containing lipids were prelabelled with myo[2-3H]inositol, the addition of either compound resulted in an increase in insulin output, an increase in 3H efflux, rapid and significant increases in labelled inositol phosphate accumulation and a sustained increase in 3H efflux after removal of the stimulant. Direct measurements of labelled inositol phosphate accumulation in islets previously stimulated with alpha-ketoisocaproate demonstrate that this sustained increase in 3H efflux was the result of a persistent increase in phosphoinositide hydrolysis and was not simply a consequence of the hydrolysis of preformed inositol phosphates into more membrane permeable species. Prior exposure of islets to alpha-ketoisocaproate or leucine also resulted in an amplified secretory response to a subsequent glucose (10 mmol/l) stimulus. While peak first phase insulin release averaged 66 +/- 4 (mean +/- SEM, n = 18) pg.islet-1. min-1 from control islets, this value increased to 204 +/- 14 and 246 +/- 11 pg.islet-1.min-1 in the leucine or alpha-keto-isocaproate pretreated islets respectively. The duration of this amplified response paralleled the duration of the persistent increase in 3H efflux. Prior alpha-ketoisocaproate exposure also amplified the subsequent insulin secretory response to tolbutamide and glyceraldehyde.(ABSTRACT TRUNCATED AT 250 WORDS)

Induction of memory in rat pancreatic islets by tolbutamide. Dependence on ambient glucose level, calcium, and phosphoinositide hydrolysis.

The ability of the sulfonylurea tolbutamide to induce insulin output, increase phosphoinositide (PI) hydrolysis, and modulate the insulin response to other agonists was assessed. At 200 microM, tolbutamide increased both insulin release and the efflux of 3H from [3H]inositol-prelabeled islets only in the presence of 5.5 or 7 mM glucose. When the glucose level was maintained at 2.75 mM, tolbutamide (200 microM) had no positive impact on either parameter. The calcium-influx inhibitor nitrendipine (200 nM) blocked the effects of 200 microM tolbutamide (with 7 mM glucose) on 3H efflux and insulin output. Prior exposure of islets to tolbutamide (200 microM) in the presence of 7 mM glucose amplified their subsequent insulin response to 10 mM glucose and 5 mM glyceraldehyde. The effect of 200 microM tolbutamide (with 7 mM glucose) was blocked by nitrendipine. Furthermore, the effect of 200 microM tolbutamide was not observed with 2.75 mM glucose; however, if the level of tolbutamide was increased to 1 mM, both PI hydrolysis and potentiated release to subsequent stimulation with 10 mM glucose were observed. Tolbutamide (200 microM with 7 mM glucose) stimulation for 20 min resulted in an increase in 3H efflux from [3H]inositol-prelabeled islets. Despite the rapid fall in insulin secretion, elevated rates of 3H efflux persisted long after the removal of the sulfonylurea from the medium. The duration of the 3H-efflux response paralleled the duration of potentiation.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of phosphoinositide metabolism in induction of memory in isolated perifused rat islets.

Prior exposure of isolated perifused rat islets to 20 mM glucose or 10 mM glyceraldehyde amplifies their subsequent insulin secretory response to 10 mM glucose. The involvement of phosphoinositide turnover in the induction of this "time-dependent potentiation" (TDP) was investigated. In islets in which inositol-containing phospholipids were prelabeled with myo-[2-3H]inositol, the addition of 20 mM glucose augments the efflux of 3H. This effect persists for approximately 50 min after the cessation of stimulation. Direct measurements of labeled inositol phosphate accumulation confirmed that this increase in 3H efflux is primarily the result of a persistent increase in phosphoinositide (PI) hydrolysis and not due to the slow efflux and/or degradation of performed [3H]inositol phosphates. The duration of the increase in 3H efflux parallels the duration of TDP. Mannoheptulose abolishes both the increase in 3H efflux evoked by 20 mM glucose and TDP. The omission of extracellular calcium plus 0.5 mM ethylene glycol-bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid also abolishes both of these effects of high glucose. D-Glyceraldehyde (10 mM) addition to 3H-inositol-prelabeled islets results in an acute efflux of 3H, a persistent efflux after removal of the D-glyceraldehyde from the perifusion medium, and the induction of TDP. Similar to the results obtained with high glucose, the return of 3H efflux rates to prestimulatory values is accompanied by the abolition of TDP. These results suggest that events associated with persistent stimulant-induced increases in phosphoinositide hydrolysis may participate in the induction and maintenance of TDP.

Cholecystokinin-induced alterations in beta-cell sensitivity. Duration, specificity, and involvement of phosphoinositide metabolism.

Prior exposure of isolated perifused rat islets to the sulfated gut hormone cholecystokinin-8 (CCK-8S) dramatically increased their insulin secretory response to 7.5 mM glucose, 10 mM arginine, and 10 mM alpha-ketoisocaproate. In the case of glucose, the heightened secretory response was still apparent 60-80 min after CCK-8S removal from the perifusion medium. Prior exposure of perifused islets to arginine (10 mM), tolbutamide (25 microM), or forskolin (1.0 microM) did not sensitize them to 7.5 mM glucose. CCK-8S exposure increased 3H efflux from islets prelabeled with [3H]inositol, and the increase in 3H efflux was sustained after CCK-8S removal from the perifusion medium. The duration of this increase in 3H efflux paralleled the temporal characteristics of this sensitization process and was significantly attenuated by 25 microM asperlicin, a competitive antagonist of CCK binding to its membrane receptor. Arginine, tolbutamide, or forskolin treatment of islets did not increase 3H efflux from [3H]inositol-prelabeled islets. The results suggest that the turnover of membrane phosphoinositides induced by CCK-8S is largely responsible for this heightened state of secretory responsiveness to various stimulants. Second-messenger molecules generated during phosphoinositide turnover may be responsible for the phenomenon of sensitization displayed by islet tissue to CCK-8S addition.

Purinergic and non-purinergic innervation in the cerebral arteries of the dog.

1 Possible involvement of sympathetic purinergic transmission in the neurogenic response of dog cerebral and basilar arteries was examined with the use of alpha, beta-methylene ATP and adrenoceptor, cholinoceptor blocking agents. 2 In the isolated basilar arteries, electrical transmural stimulation produced a transient contraction which was frequently followed by a relaxation. This transient contraction was abolished after desensitization of P2-purinoceptors with alpha, beta-methylene ATP or by treatment with guanethidine. The relaxant response induced by electrical stimulation was also attenuated but was not abolished by such treatments. Prazosin, propranolol and atropine had no significant effect on the responses to electrical stimulation. Yohimbine augmented both the contractile and relaxant responses. 3 In most preparations of the dog middle cerebral arteries, electrical transmural stimulation produced only a relaxation. This relaxation was little affected after treatment with alpha, beta-methylene ATP or guanethidine, and was not inhibited by the other adrenoceptor and cholinoceptor blocking agents. 4 Tetrodotoxin abolished the responses induced by electrical transmural stimulation in both the basilar and middle cerebral arteries. 5 Exogenous ATP (10(-6) and 10(-5)M) produced a transient contraction followed by a relaxation of the basilar arteries and a relaxation of the middle cerebral arteries. Desensitization of P2-purinoceptors abolished the contractile response to ATP without affecting the amplitude of relaxation. 6. In the basilar and middle cerebral arteries preincubated with [3H]-noradrenaline, electrical transmural stimulation evoked an increase in 3H-efflux and this response was markedly inhibited by guanethidine or tetrodotoxin but was not affected by alpha, beta-methylene ATP. Yohimbine increased the evoked 3H-efflux. 7. These findings indicate that cerebral arteries of the dog are innervated by sympathetic purinergic nerves and non-sympathetic nerves which liberate unknown vasodilator substance(s), and that the former nerves are more dominant in the neurogenic response to electrical stimulation of the dog basilar artery than in the middle cerebral artery.

Alpha 2-adrenoceptor blockade prevents cardiac glycoside-evoked neurotransmitter release from sympathetic nerves in dog saphenous vein.

1 The effect of alpha-adrenoceptor antagonists upon neurotransmitter release evoked by cardiac glycosides from sympathetic nerve terminals has been investigated in dog saphenous vein. 2 In rings of saphenous vein preloaded with [3H]-noradrenaline, acetylstrophanthidin (ACS) caused a concentration-dependent efflux of 3H (EC50 ca. 4.4 microM) that was attenuated by phentolamine and yohimbine but not by prazosin. 3 In helical strips of saphenous vein superfused with ACS at EC50 the efflux of 3H-compounds in general, and of [3H]-noradrenaline in particular, occurred after a short delay and increased with time to a maximum reached at 75 min. Phentolamine and phenoxybenzamine, but not prazosin reduced the efflux of [3H]-noradrenaline and of total 3H-compounds throughout the time-course of the ACS-evoked effect. 4 In helical strips of saphenous vein the glycoside ouabain also caused an increase in [3H]-noradrenaline and in total 3H-efflux that was attenuated by phentolamine. 5 By contrast with the above, in bovine adrenal medullary chromaffin cells, which appear to have no functional alpha-adrenoceptors, ACS caused a small, but significant increase in 3H-efflux which was not prevented by phentolamine. 6 Phentolamine, at concentrations that attenuate markedly the ouabain- or ACS-evoked increase in 3H-efflux from dog saphenous vein, did not cause significant inhibition of cocaine-sensitive [3H]-noradrenaline uptake nor did it reduce the extent of the 3H-efflux evoked either by tyramine or by reduced extracellular Na+. These findings imply that phentolamine does not affect ACS-evoked neurotransmitter release by an action on the catecholamine uptake mechanism. 7. It is concluded that the cardiac glycoside-evoked increase in neurotransmitter release from noradrenergic nerve terminals ofdog saphenous vein is modulated by a mechanism that involves an alpha2- adrenoceptor.

Asperlicin antagonizes stimulatory effects of cholecystokinin on isolated islets.

Asperlicin, a product derived from the fungus Aspergillus alliaceus, antagonized the multiple stimulatory effects of cholecystokinin (CCK-8S) on isolated islets. At a level of 10 microM, asperlicin completely inhibited insulin release in response to 25 nM CCK-8S. Increasing the level of CCK-8S to 100 nM partially restored a secretory response, while an even greater insulin stimulatory effect was noted with 500 nM CCK-8S. The inhibitory effect of asperlicin on CCK-8S-induced release was reversible. Asperlicin exposure had no effect on glucose or glyceraldehyde-induced secretion. Asperlicin reduced, in parallel with secretion, the increase in 3H efflux from [3H]inositol prelabeled islets usually noted with CCK-8S addition. Asperlicin did not influence the small glucose-stimulated increase in 3H efflux. The results support the notion that asperlicin is a specific and potent antagonist of the multiple stimulatory effects of CCK-8S on islet tissue.

Effects of calcium antagonists on release of [3H]noradrenaline in rabbit aorta

The effects of several organic Ca antagonists and putative calmodulin antagonists on the release of [3H]noradrenaline from rabbit aorta were examined. A 65.4 mM K solution and electrical stimulation induced a Ca-dependent increase in the 3H-efflux from the adventitial layer (containing nerve terminals) but not from the media-intimal layer. Verapamil, D600, N-(6-aminohexyl)-5-chloro-l-naphthalenesulphonamide (W-7) and N2-dansyl-L-arginine-4-t-butylpiperidine amide (TI 233), in the concentration of 10−5 M, did not inhibit the increase in 3H-efflux. A high concentration (2 × 10−4 M) of verapamil induced a Ca-independent increase in the 3H-efflux from the adventitia but not from the media-intimal layer. D600, diltiazem, W-7 and TI233, in the concentration of 2 × 10−4 M, also increased the 3H-efflux; 65.4 mM K, added in the presence of 2 × 10−4 M verapamil or W-7, produced no further increase in 3H-efflux. The 3H-efflux evoked by electrical stimulation was only partially inhibited by 2 × 10−4 M verapamil. It is suggested that the evoked release of noradrenaline in rabbit aorta is relatively insensitive to organic Ca antagonists and calmodulin antagonists, and that high concentrations of these inhibitors themselves release noradrenaline by a mechanism independent of external Ca.

Pharmacological Properties of Presynaptic β-Adrenoceptors in Guinea-Pig Pulmonary Arteries

Pharmacological properties of the facilitatory presynaptic β-adrenoceptor mechanism were studied in superfused spiral preparations of guinea-pig pulmonary arteries preloaded with 3H-norepinephrine. (-)-lsoproterenol (0.3 μM)-induced increases in total 3H efflux per pulse evoked by transmural field stimulation (1, 5, 10 and 20 Hz, 10 V, 2 msec pulse width, 100 pulses and 30 min intervals) were neither dependent on impulse-frequencies nor selective at lower frequencies. Isoproterenol increased 3H efflux at 5 Hz in a concentration-dependent manner (1 nM to 1 μM): pD2 was 7.7. Salbutamol increased 3H efflux in a similar manner to isoproterenol: pD2 was 7.4. Prenalterol at 3 μM only slightly increased 3H efflux. Tazolol (10 nM to 3 μM) produced no increases. Atenolol (3 μM) and practolol (3 μM) did not antagonize isoproterenol (0.3 μM)-induced increases in 3H efflux. Butoxamine (3 μM) and H 35/25 (3 μM) did antagonize this parameter. (-)-Epinephrine (1 nM to 0.1 μM) decreased 3H efflux at 5 Hz and concentration-dependently increased this parameter in the presence of 10 μM phentolamine. (-)-Norepinephrine (10 nM to 1 μM) concentration-dependently inhibited evoked 3H efflux and did not increase the parameter in the presence of 10 μM phentolamine, 10 μM cocaine and 10 μM normetanephrine. Thus, there exist presynaptic β2-subtype receptors on noradrenergic nerve endings innervating guinea-pig pulmonary arteries.

Pharmacological properties of presynaptic beta-adrenoceptors in guinea-pig pulmonary arteries.

Pharmacological properties of the facilitatory presynaptic beta-adrenoceptor mechanism were studied in superfused spiral preparations of guinea-pig pulmonary arteries preloaded with 3H-norepinephrine. (-)-Isoproterenol (0.3 microM)-induced increases in total 3H efflux per pulse evoked by transmural field stimulation (1, 5, 10 and 20 Hz, 10 V, 2 msec pulse width, 100 pulses and 30 min intervals) were neither dependent on impulse-frequencies nor selective at lower frequencies. Isoproterenol increased 3H efflux at 5 Hz in a concentration-dependent manner (1 nM to 1 microM): pD2 was 7.7. Salbutamol increased 3H efflux in a similar manner to isoproterenol: pD2 was 7.4. Prenalterol at 3 microM only slightly increased 3H efflux. Tazolol (10 nM to 3 microM) produced no increases. Atenolol (3 microM) and practolol (3 microM) did not antagonize isoproterenol (0.3 microM)-induced increases in 3H efflux. Butoxamine (3 microM) and H 35/25 (3 microM) did antagonize this parameter. (-)-Epinephrine (1 nM to 0.1 microM) decreased 3H efflux at 5 Hz and concentration-dependently increased this parameter in the presence of 10 microM phentolamine. (-)-Norepinephrine (10 nM to 1 microM) concentration-dependently inhibited evoked 3H efflux and did not increase the parameter in the presence of 10 microM phentolamine, 10 microM cocaine and 10 microM normetanephrine. Thus, there exist presynaptic beta 2-subtype receptors on noradrenergic nerve endings innervating guinea-pig pulmonary arteries.

Effects of nerve stimulation on enzyme secretion from the in vitro rat pancreas and 3H-release after preincubation with catecholamines.

In the presence of the cholinergic antagonist atropine, electrical field stimulation (FS) (5-20 Hz) caused a marked, reversible increase in the amylase output from superfused rat pancreatic segments. Adrenaline and noradrenaline evoked dose-dependent increases in amylase output which were similar to those produced by FS. The FS- and catecholamine-evoked amylase secretions were abolished by the beta-adrenergic antagonist propranolol. The FS-evoked secretion could be abolished by either the removal of external Ca2+ or the application of tetrodotoxin (TTX, 2 X 10(-6) M). FS also resulted in a reversible increase in the fractional efflux of tritium (3H) from rat pancreatic tissues preincubated with either 3H-noradrenaline or 3H-adrenaline. The effects of FS (5-20 Hz) on 3H efflux were abolished by TTX (2 X 10(-6) M). TTX had no effect on the enhancement of 3H efflux caused by elevation of external potassium concentration (high K+, 75 mM). Removal of superfusate Ca2+ completely abolished both the FS- and high K+-induced increases in 3H efflux. These observations suggest that intrinsic nerve stimulation (i.e. FS) results in the Ca2+-dependent release of sympathetic neurotransmitter, noradrenaline, which has a direct secretory action on the rat pancreas. Furthermore, the findings suggest that adrenaline can be taken up by nervous elements. This raises the possibility that uptake and re-release of circulating adrenaline might contribute to the control of rat pancreatic enzyme secretion by the adrenergic nervous system.

Effects of cinepazide on the purinergic responses in the dog cerebral artery.

The effects of cinepazide, 1-[(1-pyrrolidinylcarbonyl)methyl]-4-(3,4,5-trimethoxycinnamoyl )piperazine hydrogen maleate, were studied in isolated dog cerebral arteries. Cinepazide in concentrations ranging from 10(-6) to 10(-5) M augmented the relaxing responses to ATP, adenosine and cAMP. However, this agent did not affect the relaxations induced by isoproterenol and papaverine and the contractions induced by 5-HT, prostaglandin F2 alpha and ATP. In the basilar artery preloaded with 3H-norepinephrine or 3H-adenosine, electrical transmural stimulation resulted in a marked increase in 3H-efflux. This efflux accompanied an initial transient contraction followed by a relaxation. Cinepazide slightly reduced the 3H-efflux evoked by electrical stimulation. However, the relaxing response was mostly augmented by the treatment with cinepazide. The relaxing responses to ATP, adenosine, cAMP and electrical transmural stimulation were attenuated by theophylline. These results suggest that cinepazide selectively potentiates the relaxing response mediated through purinergic P1-receptors.

Further characterization of presynaptic β-adrenoceptors in guinea-pig pulmonary arteries

Presynaptic β-adrenoceptors were further characterized in spiral strips of guinea-pig pulmonary arteries preloaded with [ 3H]norepinephrine. l-Metoprolol (3 × 10 −6 M) inhibited isoproterenol (3 × 10 −7 M)-induced increases in 3H efflux by transmural field stimulation, whereas the d-isomer produced to inhibition. However, IPS 339, H 35 25 , butoxamine and metoprolol (3 × 10 −6 M) antagonized salbutamol (3 × 10 −7 M)-induced increases in the parameter, whereas acebutolol, bevantolol and practolol (3 × 10 −6 M) produced no antagonism. Presynaptic β-adrenoceptors in guinea-pig pulmonary arteries appear to have characteristics similar to those postsynaptic classical β-adrenoceptors.

Role of Ca2+ in secretagogue-stimulated breakdown of phosphatidylinositol in rat pancreatic islets.

Breakdown of phosphatidylinositol (PI) has been shown to be increased during Ca2+-mediated stimulation of cellular responses in many systems and has been proposed to be involved in stimulus-secretion coupling. The effects on PI breakdown of insulin secretagogues that alter cellular Ca2+ or cyclic (c)AMP levels were investigated in perifused rat islets of Langerhans. Isolated islets were labeled with myo-[2-3H(N)]inositol and the efflux of 3H-labeled metabolites was monitored. Glucose (16.7 mM) greatly increased 3H release in a manner that paralleled the second phase of the insulin secretory response; by 60 min, the amount of [3H]PI in the islet decreased by 50%. Removal of Ca2+ from the perifusate or blockade of Ca2+ entry through the voltage-dependent channels by D600 (20 microM) abolished the glucose-induced increase in 3H efflux. Depolarization with 47 mM K+, which increases Ca2+ entry, stimulated protracted 3H and insulin release. Glucose-stimulated output of 3H was not prevented by epinephrine (1 microM) even though the insulin response was abolished. In contrast, 3H output was not affected by isobutylmethylxanthine (1 mM), known to raise cellular levels of cAMP, although insulin release was stimulated. These findings indicate that PI breakdown is not related to the exocytotic process since stimulation of insulin release and PI breakdown could be uncoupled, and that it is not associated with cAMP-mediated regulation of insulin release. PI breakdown in islets differs from the immediate, transient phenomenon reported in other systems in both its timing and requirement for Ca2+. It appears to result from the entry of Ca2+ and not to be the mechanism by which glucose initiates Ca2+ influx.