Pharmacological characteristics of the positive inotropic effect of angiotensin II in the rabbit ventricular myocardium.

1. In order to elucidate the mechanism underlying the positive inotropic effect (PIE) of angiotensin II (AII), we measured changes in phosphoinositide hydrolysis and contractile force induced by AII in the rabbit ventricular myocardium. 2. AII (1.0 nM-3 microM) produced a PIE in a concentration-dependent manner in the presence of bupranolol (0.3 microM) and prazosin (0.1 microM), the maximal response being about 40% of that to isoprenaline and the EC50 30 nM. 3. The PIE of AII was associated with a concentration-dependent increase in the total duration of contraction; the time to peak force and the relaxation time were prolonged. 4. AII (10 nM-30 microM) elicited an accumulation of [3H]-inositol monophosphate in a concentration-dependent manner in rabbit ventricular slices prelabelled with myo-[3H]-inositol. 5. The PIE and the accumulation of [3H]-inositol monophosphate induced by AII were inhibited by a non-selective AII receptor antagonist, saralasin (10 nM-1 microM) and by a selective AT1 receptor antagonist, losartan (10 nM-1 microM), but not a selective AT2 receptor antagonist, PD 123319 (1 microM). 6. A tumour-promoting phorbol ester, phorbol 12,13-dibutyrate (PDBu, 10-100 nM), inhibited the AII-induced PIE and [3H]-inositol monophosphate accumulation in a concentration-dependent manner. 7. These results suggest that AII exerts a PIE through activation of AT1 receptors and subsequent acceleration of phosphoinositide hydrolysis. Activation of protein kinase C by PDBu may inhibit the AII-induced stimulation of phosphoinositide hydrolysis and thereby the PIE of AII in the rabbit ventricular myocardium.     

Characterization of histamine receptors modulating inotropic and biochemical activities in rabbit left atria.

The experiments were performed to identify histamine H1- and H2-receptors in rabbit left atrium and to characterize the pharmacological properties mediated by the respective subtypes of histamine receptors. High-affinity saturable binding to the left atrial membranes was obtained for [3H]mepyramine, yielding a maximum binding capacity (Bmax) of 96 fmol/mg of protein and an equilibrium dissociation constant (KD) of 3.8 nM and also for [3H]tiotidine, yielding a Bmax of 126 fmol/mg of protein and a KD of 14.7 nM. In isolated left atrium, histamine produced a concentration-dependent positive inotropic effect, an effect which was competitively antagonized by cimetidine but not altered by chlorpheniramine. Schild analysis showed that the pA2 value for cimetidine was 6.55 and the slope was not significantly different from unity. An excellent correlation was found between the increase in force of contraction and cyclic AMP in the presence of histamine, suggesting that the positive inotropic effect of histamine in rabbit left atrium is dependent on an increased level of intracellular cyclic AMP through stimulation of histamine H2-receptors. Histamine also produced concentration-dependent stimulation of phosphoinositide hydrolysis as measured by [3H]inositol monophosphate accumulation. The phosphoinositide response to histamine was blocked by chlorpheniramine and mepyramine but not by cimetidine. The data indicate that histamine H1-receptors, in addition to histamine H2-receptors, are present in the rabbit left atrium. Although this tissue lacks an inotropic response to histamine H1-receptor stimulation, the histamine H1-receptors interact with histamine to mediate the stimulation of phosphoinositide hydrolysis.     

Species-related differences in inotropic effects of angiotensin II in mammalian ventricular muscle: receptors, subtypes and phosphoinositide hydrolysis.

1. Experiments were carried out to clarify the mechanisms responsible for variations in the positive inotropic effect (PIE) of angiotensin II (AII) on ventricular muscles from various mammals. We examined the density of AII receptors, the relative proportions of receptor subtypes and the acceleration of the hydrolysis of phosphoinositide that was induced by AII, as well as the PIE of AII in ventricular muscles from the rabbit, dog, rat and ferret. 2. In the rabbit, AII (1 microM) in the presence of bupranolol (0.3 microM) and prazosin (0.1 microM) elicited a concentration-dependent PIE, which was antagonized by a selective AT1 subtype antagonist, losartan, but not by an AT2 antagonist, PD123319. AII did not have any inotropic effects in ventricular muscles from the dog, rat and ferret. 3. Specific high-affinity binding of [125I]-AII, with a similar Kd value in each case (1-2 nM), was observed with membrane fractions derived from ventricular muscle of all four species tested. 4. In the rabbit, losartan and PD123319 each displaced approximately 50% of [125I]-AII specific binding having high affinity for the receptors, and indicating that AT1 and AT2 subtypes were present in equal numbers. In the other species the AT1 subtype of receptors was predominant. 5. In all four species AII caused a concentration-dependent acceleration of the hydrolysis of phosphoinositide in ventricular slices that had been prelabelled with myo-[3H]-inositol. 6. The results indicate that the signal-transduction process distal to acceleration of the hydrolysis of phosphoinositide may be responsible for the wide range of species variations in the inotropic action of AII on mammalian ventricular myocardium.     

Potent inhibitory action of chlorethylclonidine on the positive inotropic effect and phosphoinositide hydrolysis mediated via myocardial alpha1-adrenoceptors in the rabbit ventricular myocardium

Summary The influence of the alpha_lb-adrenoceptor-selective antagonist chlorethylclonidine on the alpha₁-adrenergic positive inotropic effect and the phosphoinositide hydrolysis induced by phenylephrine was investigated in the rabbit ventricular myocardium. Pretreatment of membrane fractions derived from the rabbit ventricular muscle with 10^–5 mol/l chlorethylclonidine decreased the specific binding of [³H]prazosin (at a saturating concentration of 10^–9 mol/l) from the control value of 11.27±0.48 to 4.18±1.87 fmol/mg protein. The inhibition by adrenaline of the binding of [³H]prazosin (slope factor and affinity) was not affected by chlorethylclonidine. The positive inotropic effect of phenylephrine (in the presence of 3 × 10^–7 mol/l bupranolol) was inhibited by chlorethylclonidine in a concentration-dependent manner (10^–7–10^–5 mol/l) and abolished by 10^–5 mol/l chlorethylclonidine. The concentration of chlorethylclonidine to inhibit the phenylephrine-induced maximum response to 50% was 2.4 × 10^–6 mol/l. The accumulation of [³H]inositol monophosphate and [³H]inositol trisphosphate induced by 10^–5 mol/l phenylephrine was inhibited by chlorethylclonidine in the same concentration range. These findings indicate that the myocardial alpha₁-adrenoceptors mediating a positive inotropic effect in the rabbit ventricular myocardium may belong to the chlorethylclonidine-sensitive alpha_1b-subtype, and that the subcellular mechanism of action involve phosphoinositide hydrolysis. Send offprint requests to M. Endoh at the above address     

Inhibition of inositol phospholipid synthesis and norepinephrine-stimulated hydrolysis in rat brain slices by excitatory amino acids

Inhibition by excitatory amino acid agonists of norepinephrine (NE)-stimulated phosphoinositide hydrolysis was studied in rat brain slices. Inhibition was not observed in cortical slices prelabeled with [3H]inositol but was observed when slices were incubated simultaneously with [3H]inositol, glutamate, and NE. Therefore, we hypothesized that glutamate inhibits the synthesis of inositol phospholipids available to the alpha 1-adrenergic receptor, thereby reducing NE-stimulated phosphoinositide hydrolysis. To test this hypothesis, the distribution of [3H]inositol in cortical slices was measured after 5, 10, 20, 40 and 60 min of incubation, with some slices being exposed to 200 microM NE, 1 mM glutamate, 1 mM N-methyl-D-aspartate (NMDA), 1 mM kainate, 1 mM quisqualate, or to NE in the presence of each of the excitatory amino acid agonists. Glutamate had little effect on the slice content of free [3H]inositol, but it severely reduced the synthesis of [3H]inositol phospholipids, in the presence or absence of NE. Glutamate also abolished NE-induced production of [3H]inositol monophosphate, [3H]inositol bisphosphate and [3H]inositol trisphosphate. Quisqualate mimicked the effects of glutamate, whereas NMDA and kainate caused less inhibition of the synthesis of [3H]inositol phospholipids and did not inhibit the response to NE. Glutamate produced similar inhibitory effects in slices from hippocampus and striatum. To test if the inhibitory effect of glutamate was the result of irreversible cell damage, cortical slices were incubated with 1 mM glutamate for 60 min prior to exposure to [3H]inositol and NE. Preincubation with glutamate did not reduce the synthesis of [3H]inositol phospholipids or inhibit NE-stimulated [3H]inositol monophosphate production. These results indicate that glutamate impairs the synthesis of inositol phospholipids. Each of the excitatory amino acid agonists, quisqualate, NMDA and kainate, inhibited [3H]inositol phospholipid synthesis, but only quisqualate affected [3H]inositol phospholipids available to the alpha 1-adrenergic receptor.     

Further evidence against the coupling of dopamine receptors to phosphoinositide hydrolysis in rat striatum

The effects of D1 and D2 dopamine receptor agonists on phosphoinositide hydrolysis were studied by measuring the accumulation of radioactive inositol phosphates in slices of rat corpus striatum prelabelled with [3H]inositol. All assays were performed in the presence of lithium. Neither the D1 receptor agonist SKF 38393 nor the D2 receptor agonist quinpirole, alone or in combination, had an effect on basal accumulation of inositol phosphates. The muscarinic receptor agonist carbachol produced a robust increase in the accumulation of inositol monophosphate and a smaller increase in the accumulation of inositol bisphosphate. These effects were not altered by the presence of quinpirole. Additionally, quinpirole also had no effect when assays were conducted in the presence of the muscarinic receptor antagonist scopolamine, the glutamic acid receptor antagonist kynurenic acid, and the antioxidant glutathione. These results are discussed in relation to recent contradictory reports and lend support to the position that D2 dopamine receptors are not coupled to phosphoinositide hydrolysis in rat striatum.     

Elevation of cytoplasmic calcium concentration stimulates hydrolysis of phosphatidylinositol bisphosphate in chick heart cells: effect of sodium channel activators

The sodium channel activators veratridine and batrachotoxin, the sodium ionophore gramicidin, and the calcium ionophore ionomycin stimulated phosphoinositide breakdown, as indicated by the increased accumulation of [3H]inositol monophosphate in embryonic chick heart cells. The levels of [3H]inositol trisphosphate and [3H]inositol bisphosphate were also increased by veratridine, indicating that there was increased hydrolysis of phosphatidylinositol bisphosphate by phospholipase C. The response to veratridine required both extracellular sodium and calcium, suggesting that calcium entry via Na/Ca exchange might activate phospholipase C. Fluorescence measurements with fura-2 confirmed that the sodium agents greatly increased the cytoplasmic calcium concentration. Veratridine (100 microM) increased cytoplasmic calcium from 94 +/- 4 nM to 862 +/- 103 nM, giving a maximal calcium increase in about 2 min. Batrachotoxin (1 microM) induced an even greater increase in calcium but required a longer time. Gramicidin also induced a large increase in cytoplasmic calcium which was maximal within 0.5 min. To directly test the calcium dependency of phospholipase C, we permeabilized the chick heart cells with saponin and monitored the production of inositol phosphates at different calcium concentrations. Raising the calcium concentration from 3 to 1000 nM increased the accumulation of [3H]inositol phosphates by nearly 4-fold with a half-maximal effect at about 200 nM calcium. The guanine nucleotide guanosine-5'-O-(3-thio)triphosphate (GTP gamma S) also stimulated accumulation of the InsPs and the response to (GTP gamma S) was potentiated by increasing the calcium concentration. The data suggest that the effect of the sodium agents on phosphoinositide hydrolysis results from an elevation of intracellular calcium which increases GTP-dependent phospholipase C activity. Thus, drugs or other conditions that elevate cytoplasmic calcium in heart cells may increase the hydrolysis of membrane phosphoinositides.     

Physostigmine stimulates phosphoinositide breakdown in the rat neostriatum

The reversible acetylcholinesterase inhibitor, physostigmine, stimulated in a dose-dependent manner the accumulation of [3H]inositol monophosphate ([3H]IP1) in lithium-treated neostriatal slices. The muscarinic agonists, carbachol and oxotremorine, also stimulated [3H]IP1 accumulation. Atropine completely blocked the physostigmine-induced accumulation but had no effect on the basal accumulation. Tetrodotoxin partially inhibited the physostigmine-induced [3H]IP1 accumulation but had no effect on the carbachol-induced accumulation. 4-Aminopyridine stimulated the basal [3H]IP1 accumulation and potentiated the physostigmine-induced accumulation. This potentiation was blocked by tetrodotoxin. The physostigmine dose-response curve for the stimulation of [3H]IP1 accumulation was similar to its dose-response curve to inhibit acetylcholinesterase activity in the neostriatum. The results suggest that, under our experimental conditions, the acetylcholine released spontaneously from intrinsic cholinergic neurons does not activate the striatal muscarinic receptors coupled to phosphoinositide breakdown unless the intrinsic acetylcholinesterases are inhibited.     

Inotropic changes induced by fluoroaluminates in rabbit left atrial muscles: possible involvement of G proteins.

1. The effects of fluoroaluminate complexes (NaF plus AlCl3) on force of contraction, cyclic AMP accumulation and phosphoinositide hydrolysis were examined in rabbit left atrial muscles. 2. Fluoroaluminates (1-10 mM NaF + 10 microM AlCl3) produced a biphasic inotropic response which was composed of an early small decline and subsequent increase in force of contraction. In the presence of the Al3+ chelator, deferoxamine (100 microM), the positive inotropic response was completely abolished and a sustained negative inotropic response appeared, suggesting that only the positive inotropic response is due to the action of fluoroaluminates. 3. The positive inotropic effect of fluoroaluminates was associated with a significant increase in the total duration of a single contraction; the time to peak tension and relaxation time were prolonged. In contrast, these parameters were substantially abbreviated by isoprenaline or histamine. 4. When force of contraction was increased by isoprenaline or histamine, the addition of fluoroaluminates caused a marked negative inotropic effect, which was eliminated by pretreatment with pertussis toxin. 5. Fluoroaluminates did not cause a significant increase in cyclic AMP content at concentrations of NaF in the range of 1-10 mM. However, the content of cyclic AMP was greatly elevated by fluoroaluminates when the atrial muscles were pretreated with pertussis toxin. 6. Accumulation of [3H]-inositol monophosphate in atrial muscle strips prelabelled with myo-[3H]-inositol was significantly increased by fluoroaluminates at concentrations of NaF over 1 mM. The phosphoinositide response to fluoroaluminates remained unchanged with pertussis toxin pretreatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Receptor-mediated inositol phospholipid hydrolysis in astrocytes

Astrocyte-enriched cultures of the neonatal rat cortex were incubated for 24 h with [3H]inositol to prelabel the membrane inositol phospholipids. Exposure of the cultures to either noradrenaline or carbachol in the presence of Li+ produced a time- and dose-dependent accumulation of intracellular [3H]inositol phosphates. The separation of the individual inositol phosphates formed in response to receptor stimulation revealed that the major 3H-metabolite accumulated under these conditions was inositol monophosphate but that at least some of this was due to the initial formation of inositol trisphosphate. The use of selective receptor antagonists showed that noradrenaline- and carbachol-induced [3H]inositol phosphate accumulation was the result of the activation of alpha 1-adrenoceptors and muscarinic acetylcholine (probably of the M1 subtype) receptors respectively. Agonist-evoked [3H]inositol phosphate accumulation were found to be additive but the simultaneous addition of agonists and the Ca2+ ionophore A23187, which also promoted inositol phospholipid hydrolysis, was not. Agonist-induced [3H]inositol phosphate accumulation was only partially dependent on extracellular Ca2+, whilst that elicited by A23187 was entirely Ca2+-dependent. The results suggest that alpha 1-adrenoceptors and muscarinic acetylcholine receptors in these cultures are present either on the same cells and linked to separate inositol lipid pools or associated with different subpopulations of astrocytes in these cultures. Moreover, inositol lipids other than phosphatidylinositol 4,5-bisphosphate may be hydrolysed in response to agonist stimulation.     

Ipriflavone inhibits phosphoinositide hydrolysis and Ca2+ uptake in the osteoblast-like UMR-106 cells.

The mechanism of action of ipriflavone, an isoflavone derivative, was studied in the osteoblastic-like UMR-106 cell line. Ipriflavone affected both phosphoinositide hydrolysis and 45Ca2+ uptake. A repeated treatment of UMR-106 cells (once a day, for 3 days) with ipriflavone decreased, in a concentration-dependent manner, [3H]inositol monophosphate accumulation. This effect was also achieved after single addition of high concentrations of ipriflavone or 100 nM [Asu1,7]eel-calcitonin, a semi-synthetic analog of eel calcitonin. When repeatedly added to UMR-106 cells, 17 beta-estradiol produced a marked inhibition of [3H]inositol monophosphate accumulation, an effect which appeared significant only at a concentration of 1 microM and which was accompanied by a reduced incorporation of [3H]inositol into membrane phospholipids. A repeated treatment with ipriflavone reduced 45Ca2+ uptake as well. This effect was observed also after a single addition of [Asu1,7]eel-calcitonin but not following single or repeated treatment with 17 beta-estradiol. The present data indicate the osteoblast as a direct and specific target for ipriflavone and suggest that this compound may share intracellular transducing mechanisms with other antiosteoporotic hormones such as estrogen and calcitonin.     

Effect of (+)-niguldipine on myocardial alpha 1-adrenoceptors in the rabbit.

The influence of the alpha 1A-adrenoceptor subtype-selective antagonist (+)-niguldipine on the alpha 1-mediated positive inotropic effect was assessed in the isolated rabbit ventricular myocardium. (+)-Niguldipine displaced the specific binding of [3H]prazosin to a membrane fraction derived from rabbit ventricular muscle with high (Ki = 64.6 pmol/l; RH = 23%) and low (Ki = 7.08 nmol/l) affinity. (+)-Niguldipine displaced specific [3H]CGP-121177 binding only at very high concentrations (Ki = 118 nmol/l). (+)-Niguldipine at 0.1 pmol/l and higher shifted the concentration-response curve for the alpha 1-mediated positive inotropic effect downwards, but at higher concentrations (up to 10 and 100 nmol/l) it did not cause a further shift of the curve. (+)-Niguldipine (1-100 nmol/l) did not affect the beta-mediated positive inotropic effect and the basal force of concentration. (-)-Niguldipine also showed a selective inhibitory action on the alpha 1-adrenoceptor-mediated positive inotropic effect, but its affinity and potency were approximately 1-2 log units lower than those of (+)-niguldipine. The present results indicate that the alpha 1A-adrenoceptor subtype is involved in the alpha 1-mediated positive inotropic effect. (+)-Niguldipine (or (-)-niguldipine with lower affinity) is able to antagonize selectively the cardiac alpha 1A-adrenoceptor-mediated positive inotropic effect. The magnitude of the alpha 1A-mediated inotropic effect, however, may be much less than that mediated by the alpha 1B-subtype in the rabbit ventricular myocardium.     

On the inotropic actions of arginine vasopressin in ventricular muscle of the guinea pig heart

Arginine vasopressin (AVP) (0.03-3 microM) produced transient negative responses that were followed by slowly developing positive inotropic responses in guinea pig papillary muscles. The inotropic responses were antagonized by OPC-21268 (10 microM), but not by OPC-31260, and suppressed by neomycin (3 mM). The negative inotropic response was hardly affected by staurosporine (10 nM) and H-7 (10 microM). AVP (1 microM) elicited a transient decrease followed by a slowly developing increase in I(Ca). The latter increase in I(Ca) was not elicited in the presence of staurosporine (1 nM). These results indicate that AVP stimulates V1 receptors that couple to phosphoinositide hydrolysis and thereby increases intracellular Ca2+ and activates protein kinase C.     

Histamine-induced hydrolysis of polyphosphoinositides in guinea-pig ileum and brain

The effect of histamine and the H1-selective agonist, 2-pyridylethylamine, on the accumulation of inositol monophosphate (InsP), inositol bisphosphate (InsP2) and inositol trisphosphate (InsP3) has been examined in lithium-treated slices of guinea-pig cerebellum and ileal smooth muscle. Following 45 min incubation, histamine produced a large accumulation of [3H]InsP and a smaller accumulation of [3H]InsP2 and [3H]InsP3 in both tissues. In cerebellar slices all three responses to histamine were potently and competitively inhibited by the selective H1-receptor antagonist, mepyramine. In contrast, incubation of ileal slices with mepyramine (0.1 microM) produced only a small reduction (circa 20%) in the maximal accumulation elicited by histamine of each [3H]inositol phosphate with no significant effect on the EC50 or Hill coefficient. However, when 2-pyridylethylamine, instead of histamine, was used to stimulate inositol phospholipid hydrolysis in ileal smooth muscle, the agonist-induced responses appeared to be competitively antagonised by mepyramine. The results presented indicate that there is an apparent dissociation between histamine-induced InsP3 accumulation and H1-receptor-mediated contractile activity in ileal smooth muscle and suggest that agonist-induced inositol phospholipid hydrolysis in this tissue may be involved in other cellular events separate from those involving calcium.     

Comparison of postnatal changes in alpha 1-adrenoceptor binding and adrenergic stimulation of phosphoinositide hydrolysis in rat cerebral cortex

Adrenergic stimulation of phosphoinositide hydrolysis is mediated by the alpha 1-adrenoceptor subtype in many tissues including the brain. We have investigated the coupling of alpha 1-adrenoceptors to phosphoinositide hydrolysis during ontogeny. Alpha 1-adrenoceptor number and affinity were measured using [3H]prazosin binding in crude membranes of cerebral cortex and compared to the ability of the adrenergic agonists norepinephrine (NE) and phenylephrine (PE) to stimulate the formation of [3H] inositol phosphates from [3H]myo-inositol in brain slices at various ages. The greatest changes in the developmental expression of both the Bmax for [3H]prazosin binding and maximal (10(-4)M) NE- or PE-stimulated [3H]inositol phosphates were observed during the period of 7-21 days of age. No changes in the KD for [3H]prazosin were observed. However, at 14 days of age the EC50 for NE but not PE stimulation of [3H]inositol phosphates was slightly but significantly lower than at later ages. To quantitatively compare these two parameters during ontogeny, data were expressed as a percentage of the adult (greater than 65 day) value. At early ages (7 and 14 days) but not at later ages (21 and 37 days) the percent expression of [3H]prazosin binding sites was significantly greater than the maximal NE-stimulated [3H]inositol phosphates. This suggests that early in neonatal development alpha 1 adrenoceptors in brain are not tightly coupled to phosphoinositide hydrolysis.     

Maitotoxin-induced phosphoinositide hydrolysis is dependent on extracellular but not intracellular Ca2+ in human astrocytoma cells

Since maitotoxin, a potent marine toxin, is known to cause not only Ca2+ influx but also phosphoinositide hydrolysis, we investigated the Ca2+ dependency of maitotoxin-induced phosphoinositide hydrolysis in 1321N1 human astrocytoma cells. Maitotoxin elicited inositol 1,4,5-trisphosphate accumulation in a time-dependent manner. In [3H]inositol-labeled cells, maitotoxin stimulated phosphoinositide hydrolysis in an extracellular Ca2+-dependent manner. Maitotoxin also caused an intracellular Ca2+ elevation, which was abolished by an intracellular Ca2+ chelater BAPTA-AM. Interestingly, maitotoxin still caused phosphoinositide hydrolysis in the BAPTA-AM-treated cells. These results indicate that maitotoxin-induced phosphoinositide hydrolysis is dependent on extracellular but not intracellular Ca2+ in 1321N1 human astrocytoma cells.     

Further characterization of the myocardial alpha-adrenoceptors mediating positive inotropic effects in the rabbit myocardium

[3H]Prazosin bound with high affinity to the membrane fraction derived from the rabbit ventricular myocardium. Oxymetazoline displaced [3H]prazosin from its binding site, did not elicit a positive inotropic effect but antagonized the positive inotropic effect of phenylephrine mediated by alpha-adrenoceptors in the presence of a beta-antagonist. Naphazoline was more potent in displacing [3H]prazosin and behaved as a weak partial agonist. YM-12617 (5-[2-[[2-(2-ethoxyphenoxy)ethyl]amino]propyl]-2- methoxybenzenesulfonamide HCl), a potent selective alpha 1-antagonist, displaced [3H]prazosin and antagonized the alpha-mediated positive inotropic effect with equal potency. Thus, a good correlation was found between the potency of alpha-antagonists to displace [3H]prazosin and their ability to antagonize the alpha-mediated positive inotropic effect. On the other hand, there was no significant correlation between the Ki and the pD2 value of the alpha-agonists (norepinephrine, epinephrine, phenylephrine and naphazoline), indicating that there is a non-linear relationship between agonist binding to myocardial alpha 1-adrenoceptors and subsequent functional changes. Myocardial alpha 1-adrenoceptors showed some pharmacological characteristics which appear to be different from those in smooth muscle tissues.     

Inhibition of receptor-coupled phosphoinositide hydrolysis by sulfur-containing amino acids in rat brain slices

Sulfur-containing amino acids were found to inhibit norepinephrine-stimulated [3H]phosphoinositide hydrolysis in rat cortical slices. Of the amino acids tested, L-cysteine was the most potent, inhibiting the response by 42 and 85% at concentrations of 50 and 500 microM respectively. L-Cystine and L-serine-O-sulfate also inhibited the response to norepinephrine, but to a lesser degree than did L-cysteine. L-Homocysteic acid slightly potentiated phosphoinositide hydrolysis at a concentration of 100 microM, but caused inhibition at 500 microM. L-Cysteine sulfinate produced effects intermediate to those of L-cysteine and L-homocysteic acid, having no effect on the response to norepinephrine at 50 microM, but causing 84% inhibition at 500 microM. The D-isomers of cysteine and homocysteic acid were much less potent than were the L-isomers. Examination of the time course of the inhibition of norepinephrine-stimulated [3H]phosphoinositide hydrolysis by L-cysteine showed that it was inhibited almost completely after 15, 30, 45 and 60 min of incubation. L-Cysteine and L-homocysteic acid caused similarly strong inhibitions of the production of [3H]inositol monophosphate, [3H]inositol bisphosphate and [3H]inositol trisphosphate. The hydrolysis of [3H]phosphoinositides stimulated by norepinephrine in slices from rat hippocampus and striatum were inhibited by L-cysteine to an extent similar to that occurring in cortical slices. These results demonstrate that several sulfur-containing amino acids, some of which have been proposed to be endogenous excitatory amino acid neurotransmitters, effectively modulate the response to norepinephrine of the phosphoinositide second messenger system in rat brain.     

Bimodal action of [Asu1,7]eel-calcitonin on phosphoinositide hydrolysis in cultured anterior pituitary cells.

[Asu1,7]Eel-calcitonin, a semisynthetic analog of eel-calcitonin displaying high stability and full biological activity, was used to study the effect of calcitonin on phosphoinositide turnover in cultured anterior pituitary cells. Incubation of cells with [Asu1,7]eel-calcitonin produced a slight, concentration-dependent increase in [3H]inositol monophosphate accumulation, without modifying thyrotropin-releasing hormone (TRH)-stimulated phosphoinositide hydrolysis. This effect was correlated with a stimulatory action on prolactin secretion. Conversely, a long-term preincubation with [Asu1,7]eel-calcitonin reduced basal as well as TRH-induced [3H]inositol monophosphate formation. This effect was concentration-dependent, was not due to an increase of cyclic AMP intracellular levels, and was attenuated in the presence of maximally effective concentrations of TRH. Such a long incubation in the presence of [Asu1,7]eel-calcitonin resulted in a marked inhibition of prolactin secretion. The present data confirm and extend previous findings showing an interference of calcitonin with the intracellular cascade consequent to membrane phospholipase C activation and further support a role for calcitonin in the modulation of hormone secretion at the pituitary.     

Phosphoinositide hydrolysis mediated by histamine H1-receptors in rat brain cortex

Histamine stimulated the accumulation of [3H]inositol 1-phosphate in the presence of lithium in [3H]inositol-prelabelled slices from rat brain cortex in a concentration-dependent manner, with an EC50 value of 94.7 microM. High concentrations of antagonists of histamine H2 receptors, muscarinic receptors, alpha 1-adrenoceptors and serotonin receptors did not inhibit the effect. The histamine H1-receptor antagonists mepyramine, triprolidine, promethazine, d-chlorpheniramine and the tricyclic antidepressant doxepin inhibited the response with Ki values corresponding to an interaction with histamine H1-receptors. The EC50 for the response was about three times lower than the Ki value (approximately 300 microM) for the inhibition by histamine of [3H]mepyramine binding to membranes from rat brain cortex. Partial inactivation of H1-receptors with the alkylating antagonist phenoxybenzamine resulted in similar reductions in [3H]mepyramine binding sites and in the maximal histamine-induced [3H]inositol 1-phosphate accumulation, without affecting the KD for the radioligand or the EC50 for the response. The apparent dissociation constant for histamine calculated from these experiments (KA = 92.2 microM) was not different from the EC50 value. The present results indicate that histamine-stimulated phosphoinositide hydrolysis in rat brain cortex is mediated by H1-receptors and that no receptor reserve is present.