Effects of choroid plexus peptide IIF on adenylate cyclase and 3',5'-cyclic adenosine monophosphate in adipose tissue.

Two hypophyseal lipolytic peptides, adrenocorticotropin (ACTH) and beta-melanocyte-stimulating hormone (beta-MSH), and the extrhypophyseal lipolytic peptide IIF, were compared with regard to their effects on free fatty acid production and 3',5'-cyclic adenosine monophosphate (cAMP) concentration in isolated rabbit and rat adipose tissue, and on adenylate cyclase activity in the tissue homogenates. ACTH at concentrations of 0.01 mug/ml or more increased lipolysis and cAMP levels in both tissues. beta-MSH at concentrations of 0.001 mug/ml or more increased lipolysis and cAMP in the rabbit tissue, but a concentration of 10 mug/ml did not stimulate lipolysis and did not alter nucleotide concentration in the rat tissue. Peptide IIF at 0.01 mug/ml or more stimulated lipolysis in rabbit adipose tissue and caused an accumulation of cAMP. A concentration of 100 mug/ml failed to stimulate free fatty acid production in the rat tissue and the cAMP level was also unaffected. In a medium containing 7.6 mEq/l of Mg++ and no Ca++, ACTH at 0.1 mug/ml or more stimulated adenylate cyclase activity in both rabbit and rat adipose homogenates by 6- to 12-fold. This effect was inhibited when Mg++ was replaced by Ca++, Na+ or K+. beta-MSH stimulated adenylate cyclase in rabbit, but not in rat, adipose homogenate in Mg++-containing incubation midium; again, the effect on rabbit adenylate cyclase was suppressed when Mg++ was replaced by Ca++, Na+ or K+. Peptide IIF failed to influence adenylate cyclase in the rabbit tissue homogenate in the Mg++-containing, Ca++-free medium; but when the medium contained 7.6 mEq/l of Ca++ in place of Mg++, 0.1 mug/ml or more of IIF caused a 4- to 15-fold increase in cyclase activity. IIF did not affect cyclase in the rat tissue homogenate in the presence or absence of Ca++. The data are consistent with the conclusion that extrahypophyseal lipolytic peptide IIF, as well as hypophyseal peptides ACTH and beta-MSH, accelerates lipolysis in susceptible adipocytes by stimulating adenylate cyclase to produce cAMP. The effect of IIF on cyclase requires the presence of exogenous Ca++; that of ACTH and beta-MSH requires exogenous Mg++.     

Comparative effects of divalent cations on the methylmercury-induced alterations of acetylcholine release

Bath application of methylmercury (MeHg) at the murine neuromuscular junction blocks synchronous evoked release of acetylcholine (ACh) and then increases spontaneous release of ACh effects observed electrophysiologically as cessation of EPPS, and increased MEPP frequency (MEPPf), respectively. The objectives of the present study were to test whether the effect of MeHg on spontaneous release was Ca++-specific by substituting Sr++ or Ba++ for Ca++, whether the time course of MeHg-induced block of synchronous evoked release was altered by varying Ca++ concentrations or substituting Sr++ and whether the processes involved in the decay of elevated MEPPf after repetitive stimulation (asynchronous evoked release) were altered by MeHg. MEPPf was recorded continuously from the rat hemidiaphragm using conventional methods during pretreatment with 2 mM Ca++, 2 mM Sr++ or 0.5 mM Ba++ and subsequently with the cation plus 100 microM MeHg. The time to peak MEPPf in MeHg was not different under any condition; however, peak MEPPf was lower in Sr++ solutions than in Ca++ or Ba++ solutions. EPPs were recorded from the rat hemidiaphragm cut muscle preparation during pretreatment with either 2, 4 or 8 mM Ca++ or 2 or 4 mM Sr++ and subsequently with the cation plus 100 microM MeHg. The latency to block of the EPP in 4 and 8 mM Ca++ was not significantly different from the latency in 2 mM Ca++. The latency to block in 2 or 4 mM Sr++ was also not different from that in Ca++. In addition, under all conditions EPP amplitude remained virtually unchanged from pretreatment values until block occurred after 8 to 9 min exposure to MeHg.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antinociceptive effects of lanthanum and cerium in nontolerant and morphine tolerant-dependent animals.

Intracerebroventricularly (i.vt.) administered lanthanum chloride was found to produce antinociceptive effects as measured by the mouse tail-flick and hot plate tests and the rat tail-flick test. These antinociceptive effects were reduced by peripheral administration of the narcotic antagonist naloxone or by i.vt. administration of calcium chloride. In addition, animals made tolerant to morphine were also tolerant to the effects of La+++. In morphine-dependent mice, the incidence of both abrupt and naloxone-precipitated withdrawal jumping was reduced by La+++ administration. In view of the known inhibitory effects of La+++ on Ca++ binding and movement, these findings suggest that alterations in Ca++ localization may be involved in the analgetic action of narcotic drugs as well as the development of narcotic tolerance and dependence.     

Does calcium mediate the increase in potassium permeability due to phenylephrine or angiotensin II in the liver?

Two agonists, phenylephrine and angiotensin II, which have been shown to alter K+ permeability in the liver were investigated as to the possible role of Ca++ in the K+ release response (measured as 86Rb efflux) in liver slices. Both phenylephrine and angiotensin II caused transient increases in 86Rb efflux from liver slices. For both agonists, the first in a series of responses was independent of extracellular Ca++, but Ca++ was required to obtain a subsequent response. This dependence on extracellular Ca++ for a second response was not receptor-specific suggesting that activation of either receptor elicited the release of the same cellular pool of Ca++. The cationophore, A-23187, only slightly increased 45Ca++ efflux and was without effect on 86Rb efflux. In contrast to the ionophore, phenylephrine stimulated a precipitous rise in 45Ca++ efflux. It is proposed that the liver may be similar to a number of other tissues in that Ca++ mediates changes in K+ permeability, but that the source is a bound Ca++ store, rather than the extracellular space.     

Mechanism of action of cobra cardiotoxin in the skeletal muscle.

Cobra cardiotoxin (CTX) is a potent polypeptide in inducing irreversible contracture of the chick biventer cervicis muscle. this polypeptide is about 2000 times more potent than caffeine in inducing contracture of this muscle preparation but the rate of CTX contracture is slower. Studies on the interaction on CTX with divalent cations showed that low Ca0++ (10(-31-2 mM)-Krebs' enhanced markedly while ethylene glycol bis (beta-aminoethyl ether)-N,N'-tetraacetic acid-Krebs' or high Ca++ (12 mM) inhibited completely CTX contracture; 10 mM Mg++ enhanced but 20 mM Mg++ or 10 mM Zn++ inhibited CTX contracture. Neither Na+ nor tetrodotoxin affected CTX contracture. Comparison of CTX and caffeine contracture showed that ethylenediamine tetraacetic acid (1 mM) and Mn++ (5 mM) inhibited CTX contracture completely but inhibited caffeine contracture only partially; procaine did not inhibit CTX contracture but inhibited caffeine contracture competitively; by contrast, N-ethylmaleimide inhibited CTX but not caffeine contracture. Neither caffeine nor K+ contracture was inhibited by 12 mM Ca++. CTX could induce contracture in the depolarized muscle and the muscle with T-tubule destroyed or closed, whereas K+ failed to induce contracture in the latter. Caffeine contracture was inhibited in the muscle with a previous elicitation of CTX contracture. This antagonistic effect of CTX could be prevented by calcium. Moreover, CTX increased both 45Ca++ efflux and 45Ca++ uptake. Both Zn++ (0.6 mM) and Mg++ (10 mM) but not protamine and polylysine mimicked CTX to increase 45 Ca++ uptake. Zn++ (0.6mM) was also found to be effective in replacing Ca0++ to induce CTX contracture in 10(-6) M Ca0++. CTX increased Ca and Na but decreased K contents of the muscle. The binding of radioactive iodinated CTX was inhibited not only by unlabeled CTX but also by 10mM Ca++. All of these findings suggest that CTX may affect a membrane calcium binding site and may induce contracture by releasing the membrane calcium rather than by increasing Na+ permeability of the muscle membrane.     

The contribution of nucleus reticularis paragigantocellularis and nucleus raphe magnus to the analgesia produced by systemically administered morphine,investigated with the microinjection technique

Intracerebral administration of morphine into either nucleus reticularis paragigantocellularis (NRPG) or nucleus raphe magnus (NRM) of rats produced analgesia, as measured by the tail flick test. NRPG was more sensitive to morphine and the effect was dose dependent. The narcotic antagonist naloxone blocked these analgesic effects of morphine. The effect of intracerebral injection of naloxone on the analgesia produced by systemically administered morphine was examined. Morphine was administered subcutaneously (2.86 mg/kg) and naloxone was microinjected 35 min later. Microinjection of 5 μg of naloxone into NRM was found to be more effective in reversing the analgesia produced by morphine than naloxone microinjected into more lateral sites, including NRPG. Lesions of NRPG did not attenuate the analgesia produced by systemically administered morphine, whereas lesions of NRM did attenuate this analgesia. The analgesia produced by morphine administered into NRPG was blocked by lesions of NRM. Cinanserin, a serotonergic blocker, blocked the effects of morphine microinjected into NRM but not effects of morphine injected into NRPG. Phenoxybenzamine partially blocked the effects of morphine injected into NRPG but not the effects of morphine injected into NRM. These results show that both nuclei are sensitive to morphine, exert their effects by different synaptic mechanisms and that NRPG does not make an appreciable contribution to the analgesia produced by systemically administered morphine.     

A characterization of the antinociception produced by intracerebroventricular injection of 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate in mice

There is evidence that 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) acts as an intracellular Ca++ antagonist producing decreases in free intracellular Ca++ and inhibiting many cellular processes dependent upon intracellular Ca++. Intracerebroventricularly administered TMB-8 was active in the mouse tail-flick test (ED50 = 50 micrograms), and this antinociceptive response was antagonized by naloxone (AD50 = 0.28 mg/kg s.c.), Ca++ (0.2-0.4 mumol i.c.v.) and, to a lesser degree, by ethylene glycol bis (beta-aminoethyl ether)N,N1-tetraacetic acid (0.02 and 0.06 mumol i.c.v.). TMB-8 (i.c.v.) was only marginally active in the p-phenylquinone test. The potency of TMB-8 (i.c.v.) was potentiated 10-fold in morphine-tolerant mice in the tail-flick test (ED50 = 2.5 micrograms). TMB-8 inhibited contraction of stimulated ilea (IC50 = 2.2 microM), an effect which was neither antagonized nor reversed by naloxone (1 microM). TMB-8 did not potentiate morphine, or was it potentiated by morphine, in the stimulated guinea pig ileum. Procaine, but not lidocaine showed dose-dependent activity in the tail-flick and p-phenylquinone tests (ED50 values, 136 and 83 micrograms, respectively, i.c.v.). The antinociception produced by procaine (i.c.v.) in the tail-flick test was antagonized by naloxone (AD50 = 0.4 mg/kg s.c.) Lidocaine (100 micrograms i.c.v.) produced only 30% maximum possible effect in the tail-flick tests, and was inactive in p-phenylquinone tests. Doses of lidocaine greater than 100 micrograms i.c.v. resulted in lethality of greater than 50% of the animals tested. Thus, the activity of TMB-8 resembles that of opiates in that both are antagonized by Ca++ or naloxone in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of divalent cations, lanthanum, cation chelators and an ionophore on acetylcholine antinociception.

The antinociceptive effect of intracerebroventricularly administered acetylcholine as measured in the mouse tail-flick test was reduced by intracerebroventricularly injected calcium, magnesium and manganese. Maximum antagonism of acetylcholine-induced antinociception was observed with a 1-hour calcium pretreatment. Significant reduction existed at 2- but not 4-hour pretreatment. Barium and strontium were inactive. The antinociceptive effect of acetylcholine was potentiated by lanthanum and ethylene glycol tetraacetic acid but not by ethylenediamine tetraacetic acid. The ionophore A23187 was shown to increase greatly the antagonistic effect of a low dose of calcium. The ionophore alone did not significantly alter the effect of acetylcholine. Thus, it appears that calcium must penetrate cell membranes to reduce the effect of acetylcholine. In addition to acetylcholine, it was found that the antinociceptive effects of oxotremorine and physostigmine could also be reduced by calcium. These data indicate that alterations in intracellular calcium are involved in cholinergically induced antinociception.     

Calcium-dependent stimulation of renal medullary prostaglandin synthesis by furosemide

The present study examined the actions of furosemide and other "loop" diuretics on immunoreactive prostaglandin E (iPGE) and [14C]arachidonate (AA) release in vitro in incubates of slices from rat and dog outer or inner medulla. The loop diuretics furosemide, ethacrynic acid, bumetanide and 3-benzylamino-4-phenylthio-5-sulfamoylbenzoic acid all significantly increased [14C]AA and iPGE release (1.5- to 4-fold) into the media of rat outer and inner medulla and dog outer medullary slice incubates. By contrast, equimolar concentrations of chlorothiazide and hydrochlorothiazide were without effects on these parameters. Stimulation of [14C]AA or iPGE by furosemide was abolished by exclusion of Ca++ from the incubation media or by addition of verapamil to complete media, but was not altered by exclusion of Na+. Ca++-free media or verapamil also abolished the increases in [14C]AA and iPGE induced by ionophore A23187. By contrast, these incubation conditions did not influence the iPGE responses to hypertonic mannitol or exogenous AA. The presence of Ca++-responsive acyl hydrolase activity was demonstrated in the microsomal fraction from rat outer medulla. However, this activity was not altered by addition of furosemide to the subcellular fraction in the presence or absence of Ca++. Thus, furosemide and other loop diuretics stimulate renal medullary iPGE synthesis in vitro, and may do so through Ca++-mediated or dependent enhancement of the release of AA.     

The neutralization of antibiotic action by metallic cations and iron chelators

The neutralization of the action of a variety of antibiotics on klebsiellae by moderate doses of di- and tri-valent metallic cations, was measured in vitro. Some beta-lactams tested were affected by Mg++ and by Cu++. Of six tetracyclines one was moderately neutralized by Ca++, two by Mg++, three by Cu++, and all, strongly, by Fe+++. Erythromycin was affected by Ca++, lincomycin by Ca++, Mg++ and Cu++. Aminoglycosides were affected by Ca++ and Mg++ and strongly by Fe+++. Five antibiotics (three beta-lactams and two macrolides) with high MICs for klebsiellae were tested against staphylococci: most of the reversing agents were ineffective. The microbial iron chelators, desferrioxamine and enterochelin were largely inactive, affecting only two aminoglycosides.     

Ionic and secretory response of pancreatic islet cells to minoxidil sulfate

Minoxidil sulfate is an antihypertensive agent belonging to the new class of vasodilators, the "K+ channel openers." The present study was undertaken to characterize the effects of minoxidil sulfate on ionic and secretory events in rat pancreatic islets. The drug unexpectedly provoked a concentration-dependent decrease in 86Rb outflow. This inhibitory effect was reduced in a concentration-dependent manner by glucose and tolbutamide. Minoxidil sulfate did not affect 45Ca outflow from islets perfused in the presence of extracellular Ca++ and absence or presence of glucose. However, in islets exposed to a medium deprived of extracellular Ca++, the drug provoked a rise in 45Ca outflow. Whether in the absence or presence of extracellular Ca++, minoxidil sulfate increased the cytosolic free Ca++ concentration of islet cells. Lastly, minoxidil sulfate increased the release of insulin from glucose-stimulated pancreatic islets. These results suggest that minoxidil sulfate reduces the activity of the ATP-sensitive K+ channels and promotes an intracellular translocation of Ca++. The latter change might account for the effect of the drug on the insulin-releasing process. However, the secretory response to minoxidil sulfate could also be mediated, at least in part, by a modest Ca++ entry.     

Involvement of calcium and iron in Quin 2 toxicity to isolated hepatocytes

When treated with the cytosolic Ca++ indicator Quin 2-acetoxymethyl ester (Quin 2-AM), isolated hepatocytes exhibited signs of toxicity, such as extensive lipid peroxidation and vitamin E loss and release of lactate dehydrogenase. Lipid peroxidation induced by this agent was blocked completely by cotreatment of the cells with ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid, EDTA, ruthenium red, carbonyl cyanide m-chlorophenylhydrazone, desferal and trifluoperazine, and was partially inhibited by quinacrine and indomethacin. With the exception of carbonyl cyanide m-chlorophenylhydrazone and quinacrine, these agents also inhibited lactate dehydrogenase leakage. Although the results with ruthenium red suggested that Quin 2-AM may cause toxicity by altering handling of Ca++ by mitochondria, mitochondrial membrane potential was not altered in cells treated with Quin 2-AM until after toxicity occurred. Evidence of a direct, potentiative effect of Quin 2 on iron-induced lipid peroxidation was gained from experiments with liposomes. Treatment of cells with Quin 2-AM did not enhance nitro blue tetrazolium reduction, suggesting that Quin 2 did not stimulate O2- production by the cells. Direct chelation of Ca++ did not appear to be involved in the mechanism of Quin 2 toxicity, for an analog of Quin 2 that is virtually nonhydrolyzable, which greatly limits the binding of Ca++, also caused lipid peroxidation and cell death. These results suggest that Quin 2 causes toxicity by chelating iron or by activating some cellular process(es) that is dependent on the presence of iron or Ca++.     

Calcium ion requirement for acetylcholine-stimulated breakdown of triphosphoinositide in rabbit iris smooth muscle.

Previous studies from this laboratory have established that addition of acetylcholine (ACh) or norepinephrine to 32P-labeled rabbit iris smooth muscle increases significantly the breakdown of triphosphoinositide (TPI) and that these stimulatory effects are blocked by atropine and phentolamine, respectively. The present studies were undertaken in order to show the effect of Ca++ on the ACh-stimulated breakdown of TPI ("TPI effect") in this tissue. Paired iris smooth muscles were prelabeled with 32Pi for 30 minutes at 37 degrees C in Ca++-free iso-osmotic salt medium. The prelabeled irises were then washed and incubated for 10 minutes in nonradioactive Ca++-free medium which contained 10 mM 2-deoxyglucose under various conditions. The phospholipids were isolated by means of two-dimensional thin-layer chromatography and their radioactivities were determined. In the absence of Ca++, 50 micrometer ACh increased TPI breakdown and phosphatidic acid (PA) labeling by 16 and 38%, respectively. In the absence of ACh, 0.75 micrometer Ca++ increased TPI breakdown and PA labeling by 11 and 20%, respectively. When both ACh and Ca++ were added, the increase in TPI breakdown and PA labeling rose to 32 and 74%, respectively. The labeling of phosphatidylinositol was found to be insensitive to the presence of Ca++. Ca++ was determined in the iris smooth muscle and it was found to contain 3.13 mumol of Ca++ per g of tissue. This was reduced by 80% after the muscle was washed and incubated in a medium which contained 0.25 micrometer ethyleneglycol bis (beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA). The TPI effect was abolished by 0.25 micrometer EGTA and restored when excess Ca++ (1.25 micrometer) was added. Concentrations of Ca++ as low as 50 micrometer provoked a TPI effect. Sr++ (2 micrometer), but not Ba++ or Mn++, was found to substitute partially for Ca++. Ionophore A-23187 (20 micrometer) was found to increase the breakdown of TPI and labeling of PA by 11 and 24%, respectively. High concentrations of Ca++ (20 mM) exerted similar effects. The increase in TPI breakdown and PA labeling in response to these agents, in contrast to the TPI effect in response to ACh, was not blocked by atropine. This suggests that the observed effects are not caused by the release of endogenous ACh from the muscle. A possible interpretation for the above observations on the role of Ca++ in the TPI effect at the postsynaptic membrane of the iris smooth muscle could be: (formula: see text).     

Ca++ inhibition of isoproterenol responses in mammalian skeletal muscle

In noncontracting mouse hemidiaphragms incubated in modified Krebs-Ringer--bicarbonate buffer with 10 mM Ca++, isoproterenol-stimulated phosphorylase a formation, conversion of phosphorylase kinase to the activated form, elevation of cyclic AMP-dependent protein kinase activity ratios and increase in cyclic AMP concentrations were reduced 35 to 50% over the responses in buffer with 2.5 mM Ca++. In buffer with 10 mM Ca++, the initial rate of isoproterenol-stimulated cyclic AMP accumulation was 59% of that in buffer with 2.5 mM Ca++. The inhibitory action of Ca++ on cyclic AMP accumulation was antagonized by verapamil, but not by inhibitors of cyclic nucleotide phosphodiesterase activity. In buffer with 2.5 mM Ca++, isoproterenol-stimulated cyclic AMP accumulation was inhibited by A23187 and caffeine, agents that can increase intracellular Ca++ concentrations. In addition to Ca++, high concentrations of Co++, Ni++, Mn++ and, to a lesser extent, Sr++ inhibited the isoproterenol response. The results of these studies indicate that high buffer Ca++ concentrations inhibit the response of the glycogenolytic pathway to isoproterenol by an action on cyclic AMP formation. We propose that the site of the inhibitory action of Ca++ is the divalent metal activator site associated with hormone-stimulated adenylate cyclase activity.     

Lead accumulation by rat renal brush border membrane vesicles

Pb++ accumulation by rat renal cortical brush border membrane vesicles was evaluated by in vitro incubation with rapid filtration technique. Pb++ uptake was time- and concentration-dependent, with apparent saturation of binding sites at 100 to 200 microM (5 sec initial rate experiments). Equilibrium binding studies (60-min incubation) showed that the ratio of bound Pb++ to free Pb++ was constant at 1.25 +/- 0.07 between 0.01 to 10 microM Pb, with decreasing bound to free ratios at higher concentrations. Osmotic experiments showed that Pb++ uptake was due primarily to membrane binding rather than intravesicular accumulation. Electrochemical gradients of NaCl, KCl or protons did not increase vesicle uptake of Pb++. Incubation of vesicles with a number of amino acids did not stimulate Pb++ uptake although two (cysteine and glutathione) and the chelators EDTA or ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid) completely blocked this process. Competition studies with a number of other metals (at 10 microM and 1 mM) showed that only Sn++ or Sn +, La , Fe++ or Fe and Cu++ produced significant reductions in Pb++ uptake whereas Mg++, Ca++, Zn++, Cd++ and Hg++ were without effect on this process. Release of 203Pb from preloaded vesicles was accelerated in the presence of either cysteine or Sn +. Prior in vivo exposure to Pb (3 mg of Pb/kg i.v.) reduced Pb uptake to 70% of that of vesicles prepared from control animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of morphine on calcium uptake by lysed synaptosomes.

The effect of morphine on the uptake of 45Ca++ was studied in lysed synaptosomes obtained from homogenates of whole mouse brain. The addition of morphine, 10(-6) M, to the incubation medium or acute administration of 10 or 20 mg/kg s.c. resulted in a decrease in 45Ca++ uptake; this decrease was observed only in the presence of ATP (3 mM). In contrast, after morphine pellet implantation (72 hr) to induce tolerance and physical dependence, an enhancement of lysed synaptosomal 45Ca++ uptake occurred; the increase was obtained in the presence but not in the absence of ATP. The enhancement of Ca++ uptake appears to be related with the degree of tolerance and dependence development since a linear relationship was noted between the time of morphine pellet implantation and the increase in 45Ca++ uptake by lysed synaptosomes. The acute inhibitory action on 45Ca++ uptake by morphine was prevented in vitro by naloxone, 1.9 x 10(-8) M, and in vivo by 2 mg/kg of naloxone s.c. and the chronic enhancing action of morphine by the simultaneous implantation of a naloxone pellet with the morphine pellet. The present findings lend further support to our previous reports in which we suggest that alterations in Ca++ flux may be involved with morphine analgesia, tolerance and physical dependence.     

Effects of ionophore A23187 on base-line and vasopressin-stimulated sodium transport in the toad bladder.

The cation specific ionophore A23187 (Io) is a useful tool for studying the role of intracellular Ca++ (Ca++)i in physiologic processes. The present studies explore the role of (Ca++)i on Na transport in the toad bladder. Scraped bladder cells exposed to 1 muM Io for 60 min took up 100% more 45Ca than control cells. Io, 1 muM, added to the serosal side of bladders incubated in standard Ringers containing 2.5 mM Ca++ inhibited short circuit current (SCC) values by a mean of 30% at 60 min and 50% at 90 min. Io did not inhibit SCC significantly in bladders incubated in Ringers containing 0.2 mM Ca++. These data indicate that the effects of Io on SCC depend on the levels of external Ca++ and suggest that entry of Ca++ into cells mediates the inhibition of base-line SCC. PReincubation of the bladders with either lanthanum chloride or pentobarbital prevented the increased 45Ca uptake produced by ionophore as well as theinhibition of SCC caused by the antibiotic. Vasopressin, antidiuretic hormone (ADH). 10 MU/ml, increased peak SCC by 247% in bladders preincubated for 1 h in Ringers with 2.5 mM Ca++ and 1 muM Io and by 318% in control bladders (P less than 0.01). Bladders exposed to 1 muM Io in Ringers with 0.2 mM Ca++ had an increase in SCC after ADH comparable to that observed in controls. Since the effects of ADH on SCC are mediated by cyclic AMP, we tested the effects of Io on cAMP production by scraped toad bladder cells. ADH increased cAMP from 8 to 30 pmol/mg protein in controls but it did not increase cAMP over base-line values in the presence of Io when the Ringers contained 2.5 mM Ca++. Io did not inhibit cAMP production in response to ADH when the Ca++ in the Ringers was 0.2 mM. The results indicate that Io inhibits baseline and ADH stimulated SCC by increasing (Ca++)i or Ca++ bound to the cell membrane. It is suggested that: ()( (Ca++)i or membrane-bound Ca++ plays a key role in base-line and ADH stimulated Na transport in the toad bladder; (2) inhibition of ADH stimulated SCC may be due inpart to decreased cAMP generation in response to ADH when (Ca++)i or membrane-bound Ca++ levels are increased.     

Effects of acute and chronic morphine treatments on calcium localization and binding in brain

Acute subcutaneous injection of 25 mg/kg of morphine in rats decreased synaptosomal CA++ levels by 29% without altering the Ca++ content of other subcellular fractions. In contrast, chronic morphine treatment of mice or rats by pellet implantation selectively increased synaptosomal Ca++ levels by almost 100%. This increase in Ca++ induced by morphine was blocked by simultaneous chronic administration of naloxone. The binding of low concentrations (10(-7)-10(-5) M) of 45Ca++ to synaptic plasma membranes was increased by acute morphine treatment and decreased by chronic administration. The binding of higher concentrations (10(-3) M) of 45Ca++ to synaptic vesicles was also increased by acute morphine treatment and decreased by chronic treatment. Changes in binding were not observed with other subcellular fractions. It is suggested that these highly selective changes in Ca++ levels and binding may represent mechanisms by which acute morphine treatment interferes with synaptic transmission and by which chronic administration overcomes these effects, resulting in tolerance and dependence.     

Evaluation of the discriminative effects of morphine in the rat.

The discriminative effects produced by morphine in the rat were evaluated using a two-choice, discrete trial avoidance task. Stimulus control of behavior was attained with a dose of morphine one-third to one-tenth of that used in previous studies. Morphine produced dose-related discriminative effects over a 100-fold dose range. The stimulus control produced by the discriminative effects of morphine met the following criteria for classification as a specific narcotic effect: 1) oxymorphone, levorphanol, methadone and meperidine, narcotic analgesics from diverse chemical families, also produced dose-related morphine-like discriminative effects; 2) dextrorphan and thebaine, compounds structurally related to the narcotics but lacking narcotic activity, failed to produce morphine-like discriminative effects; 3) effects were blocked by the narcotic antagonist naloxone; and 4) tolerance to the discriminative effects developed upon the repeated administration of morphine and cross-tolerance extended to methadone. The discriminative effects produced by morphine were further characterized by evaluating the capacity of prototypes of other classes of psychoactive drugs to produce morphine-like discriminative effects. Profadol and pentazocine, euphorogenic analgesics with mixed agonist and narcotic antagonist properties, produced dose-related morphine-like discriminative effects whereas cyclazocine, a dysphorogenic analgesic with mixed agonist and narcotic-antagonist properties, did not. In addition, the nonopioid psychoactive drugs d-amphetamine, pentobarbital and chlorpromazine also failed to produce morphine-like discriminative effects. Thus, morphine-like discriminative effects were produced uniquely by the narcotic analgesics and euophorogenic analgesics with mixed agonist and narcotic antagonist properties. These results suggest that the component of action of morphine that enables it to function as a discriminative stimulus in the rat is analogous to the component of action of morphine responsible for producing subjective effects in man.     

Vasodilatory action of HA1004 [N-(2-guanidinoethyl)-5-isoquinolinesulfonamide], a novel calcium antagonist with no effect on cardiac function

A novel compound, N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA1004) was found to be a potent relaxant of blood vessels. Rabbit aortic strips contracted by 18 mM KCl relaxed in the presence of HA1004, with an ED50 value of 2.2 X 10(-6) M. The isolated guinea-pig atria on right ventricular papillary muscles did not respond to HA1004, even at a concentration of 3 X 10(-4) M. HA1004, like verapamil, produced a competitive inhibition of the Ca++-induced contraction of the depolarized rabbit aorta. The pA2 value of HA1004 for the Ca++-induced contraction was 6.60. Atropine, propranolol, theophylline or indomethacin had no effect on the HA1004-induced relaxation. HA1004 (3 X 10(-6) M) inhibited the contraction produced by Ca++ ionophore, A23187, whereas verapamil or diltiazem had no effect on this contraction, even at a concentration of 3 X 10(-5) M. Moreover, HA1004 produced a competitive inhibition of Ca++-induced contraction of the A23187-treated aorta and inhibited the phenylephrine-induced contraction elicited in ca++-free solution, thereby suggesting that this drug affects intracellular rather than extracellular Ca++. The present findings indicate that HA1004 is a novel calcium antagonistic vasodilator with no effect on cardiac function and is apparently of a different class of calcium antagonist from verapamil.