Guanine nucleotide and pyrophosphate activate exogenous phosphatidylinositol 4,5-bisphosphate hydrolysis in rat liver plasma membranes.

5'-guanylylimidodiphosphate (GppNHp) in the presence of deoxycholate, stimulated the phospholipase C-mediated hydrolysis of exogenous [3H]phosphatidylinositol 4,5-bisphosphate ([3H]PIP2) to myo-[3H]inositol 1,4,5-trisphosphate in rat liver plasma membranes. Activation was not specific for guanine nucleotides as 5'-adenylylimidodiphosphate, imidodiphosphate and pyrophosphate stimulated the enzyme with similar efficacies and potencies. Enzyme activation by GppNHp was most pronounced when [3H]PIP2 was used as substrate. No added Ca++ was required for [3H]PIP2 breakdown but hydrolysis was inhibited by divalent ion chelators. GppNHp stimulation was apparent in the presence of Ca++ or Mg++ as well as chelator concentrations that partially inhibited the enzyme, indicating that this effect was not attributed to changes in affinity of these divalent cations for the enzyme or substrate. These results suggest that guanine nucleotides can stimulate the hydrolysis of exogenous [3H]PIP2 in rat liver membranes by a non-specific effect probably due to the interaction of the diphosphate moiety with the enzyme or substrate.

Guanine nucleotide regulation of [3H]vasopressin binding to liver plasma membranes and solubilized receptors. Evidence for the involvement of a guanine nucleotide regulatory protein.

A guanine nucleotide regulatory protein may be involved in vasopressin-receptor-mediated polyphosphoinositide breakdown in rat liver. Therefore we examined the effects of the non-hydrolysable guanine nucleotide guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) on [3H]vasopressin ([3H]AVP) binding to hepatic plasma membranes and detergent extracts. [3H]AVP bound to a single set of high-affinity binding sites in membranes. Addition of p[NH]ppG decreased the affinity of receptor binding without altering the maximal binding capacity. The rate of dissociation of [3H]AVP from membrane-bound receptors was also enhanced by p[NH]ppG. Solubilization of [3H]AVP-prelabelled membranes with dodecyl beta-D-maltoside resulted in a [3H]AVP-receptor complex that was unstable in solution. Incubation of these extracts for 5 min at 30 degrees C resulted in a 40% loss of bound [3H]AVP, whereas in the presence of p[NH]ppG there was a 54% loss. However, when membranes were prelabelled with [3H]AVP and p[NH]ppG and then solubilized, the resulting hormone-receptor complex was still temperature-labile but insensitive to the further addition of p[NH]ppG. The molecular size of soluble vasopressin receptors was estimated by gel filtration. The [3H]AVP-receptor complex was eluted as a single peak with an apparent molecular size of 258 kDa. However, no peak was detected when solubilized extract was made from membranes prelabelled with [3H]AVP and p[NH]ppG, suggesting that this receptor complex had dissociated during chromatography. It is possible therefore that the high-Mr complex contains the hormone, its receptor and a guanine nucleotide binding protein.

Thrombin, unlike vasopressin, appears to stimulate two distinct guanine nucleotide regulatory proteins in human platelets.

The thrombin-stimulated GTPase activity of human platelets was additive with respect to the GTPase stimulation effected by prostaglandin E1, but not with that stimulated by adrenaline, vasopressin and platelet-activating factor (PAF). Treatment of platelet membranes with pertussis toxin partially inhibited the thrombin-stimulated GTPase, but had no effect on the vasopressin-stimulated GTPase activity, whereas cholera toxin treatment had no effect on either of these stimulated GTPase activities. Thrombin, adrenaline and PAF, but not vasopressin, inhibited the adenylate cyclase activity of isolated plasma membranes through the action of Ni only, this being inhibited by pertussis toxin. It is suggested that thrombin exerts effects through both the inhibitory guanine nucleotide regulatory protein Ni and through the putative guanine nucleotide regulatory protein, Np, involved in regulating receptor-stimulated inositol phospholipid metabolism. However, vasopressin appears to exert its effects solely through the putative Np.

[Delivery after previous cesarean section]. / Porodaj nakon prethodnog carskog reza.

In a four-year period (1980-1983) there were 452 women in whom one of the previous pregnancies terminated by cesarean section. Out of them, 283 (62.6%) delivered vaginally and 169 (37.4%) by repeated cesarean section. Vacuum extractor was used in 106 deliveries (23.5%). In 254 (89.8%) parturients of the examined group and in 21 (5%) parturients of the control group the manual revision of the uterus was performed. Seven incomplete and one complete silent ruptures of the uterus occurred in the examined group, the incidence being 1.8%. Six rupture of the uterus were revealed during cesarean section. Two ruptures were detected by the manual revision of the uterus. The authors are of the opinion that the manual revision of the uterus in women with previous cesarean section is necessary and that the prophylactic use of vacuum extractor is not justified.

Platelet activating factor and U44069 stimulate a GTPase activity in human platelets which is distinct from the guanine nucleotide regulatory proteins, Ns and Ni.

Platelet-activating factor (PAF, 2-acetyl-1-alkyl-sn-glycero-3-phosphocholine) and the stable thromboxane-receptor agonist U44069 (9 alpha, 11 beta-epoxymethanoprostaglandin H2) stimulated GTPase activity in platelet membranes in a dose-dependent fashion, yielding Ka values of 12 nM and 27 nM respectively. The degree of GTPase activation elicited by these agents was found to be additive with the GTPase activation due to either the stimulatory (Ns) or inhibitory (Ni) guanine nucleotide regulatory proteins when activated by prostaglandin E1 and adrenaline (+propranolol) respectively. Treatment of membranes with either cholera or pertussis toxins, which inhibited markedly the receptor-mediated stimulation of the GTPase activities of Ns and Ni respectively, had no or only a small effect, respectively, on the GTPase activity stimulated by PAF and U44069. It is suggested that PAF and U44069, which stimulate inositol phospholipid metabolism in platelets, exert actions through a guanine nucleotide regulatory protein which is distinct from Ns and Ni.

Atypical characteristics of the beta-adrenoceptor mediating cyclic AMP generation and lipolysis in the rat adipocyte.

The characteristics of the rat epidydimal adipocyte beta-adrenoceptor have been examined using lipolysis and cyclic AMP accumulation in adipocytes as well as adenylate cyclase activity in fat cell membranes. The pA2 values corrected for binding to bovine serum albumin of the selective antagonists betaxolol (beta 1-selective) and ICI 118.551 (beta 2-selective) against noradrenaline or fenoterol-stimulated lipolysis were indicative of an atypical beta-adrenoceptor associated with the lipolytic response. Antagonism of isoprenaline-stimulated cyclic AMP accumulation in whole cells and adenylate cyclase activity in membranes yielded pA2 values to betaxolol, ICI 118.551 and (-)-propranolol, which suggested that the atypical beta-adrenoceptor was coupled to adenylate cyclase. Comparisons of the Ki values obtained in binding studies using [125I]-cyanopindolol with pA2 values obtained in adenylate cyclase experiments suggest that the typical beta 1-receptor identified with radioligand binding studies is not the only receptor site mediating stimulation of adenylate cyclase activity and lipolysis.

Irreversible antagonism of beta-adrenoceptors with para-amino-benzyl-carazolol provides further evidence for an atypical rat adipocyte beta-adrenoceptor.

Rat adipocytes possess typical beta 1 adrenoceptors that can be identified by 125I-cyanopindolol binding but the receptor mediating isoprenaline adenylate cyclase activation possesses properties quite unlike beta 1 or beta 2 receptors. Separation of these sites has been attempted using the photoaffinity antagonist para-amino-benzyl-carazolol. Preincubation of rat reticulocyte and adipocyte membranes with this agent followed by washing induced a concentration-dependent loss of specific 125I-cyanopindolol sites in both tissues, though the maximal loss was apparently greater in the reticulocyte. However, the loss of sites in both tissues induced a different effect on isoprenaline-stimulated adenylate cyclase. In the reticulocyte, the loss of specific sites was accompanied by an equivalent fall in the maximal stimulation of adenylate cyclase. In the adipocyte there were no significant effects of receptor site loss on the isoprenaline dose-response curve. It is suggested that this data supports the concept that an atypical beta-adrenoceptor, with relatively low affinity for many antagonists, mediates catecholamine-stimulated adenylate cyclase (and lipolysis) in the adipocyte.

Identification and subclassification of rat adipocyte beta-adrenoceptors using (+/-)-[125I]cyanopindolol.

The ligand binding characteristics of beta-adrenoceptors on membranes derived from rat whole fat pads and isolated adipocytes have been compared to those present on lung membranes using the specific ligand (+/-)-[125I]cyanopindolol [(125I]CYP). The equilibrium dissociation constant (KD) of [125I]CYP was similar in all three preparations, whilst the degree of stereospecificity displayed by the isomers of propranolol varied between lung and isolated adipocyte membranes. The rank order of potency in displacing [125I]CYP binding was isoprenaline greater than adrenaline greater than noradrenaline in lung and whole fat pads, suggesting an overall beta 2-receptor subtype, and isoprenaline greater than noradrenaline greater than adrenaline in isolated adipocytes, suggesting a beta 1-receptor. Pharmacological characterisation of receptor subtype with selective beta-adrenoceptor agents indicated that all three preparations contained a heterogeneous receptor population (lung 80% beta 2, 20% beta 1; whole fat pad 62% beta 2, 38% beta 1; isolated adipocyte 15% beta 2, 85% beta 1). In view of the high proportion of beta 2-receptors on whole fat pad membranes, the overall amount of beta 2-receptors on isolated adipocytes may reflect contamination from other cell types. The nature of the beta 1-receptors present on fat cells is discussed in relation to the apparently atypical beta-receptor involved in mediating the functional lipolytic response.