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Biomedical subjects

Z Selinger

Publications and source records attributed to Z Selinger.

At least 91 records · Page 5Linked to original sources
PubMed1978

Mechanism of adenylate cyclase activation through the beta-adrenergic receptor: catecholamine-induced displacement of bound GDP by GTP.

The fate of the guanyl nucleotide bound to the regulatory site of adenylate cyclase was studied on a preparation of turkey erythrocyte membranes that was incubated with [3H]GTP plus isoproterenol and subsequently washed to remove hormone and free guanyl nucleotide. Further incubation of this preparation in the presence of beta-adrenergic agonists resulted in the release from the membrane of tritiated nucleotide, identified as [3H]GDP. The catecholamine-induced release of [3H]GDP was increased 2 to 3 times in the presence of the unlabeled guanyl nucleotides GTP, guanosine 5'-(beta,gamma-imino)triphosphate [gpp(NH)p], GDP, and GMP, whereas adenine nucleotides had little effect. In the presence of Gpp(NH)p, isoproterenol induced the release of [3H]GDP and the activation of adenylate cyclase, both effects following similar time courses. The findings indicate that the inactive adenylate cyclase possesses tightly bound (GDP, produced by the hydrolysis of GTP at the regulatory site. The hormone stimulates adenylate cyclase activity by inducing an "opening" of the guanyl nucleotide site, resulting in dissociation of the bound GDP and binding of the activating guanosine triphosphate.

Adenylyl Cyclases↗
PubMed1978

Morphological changes in rat pancreatic slices associated with inhibition of enzyme secretion by high concentrations of secretagogues.

Stimulation of enzyme secretion in rat pancreatic slices by cholinergic agonists or by cholecystokinin-pancreozymin (CCK-PZ) and its peptide analogs showed a biphasic dose response curve. The optimal concentrations eliciting an efficient rate of enzyme secretion were 1 microM for carbamylcholine or acetylcholine, and 5 nM and 20 nM for CCK-PZ octapeptide and CCK-PZ, respectively. At higher concentrations of secretagogues, however, the rate of secretion progressively declined, and almost complete inhibition was achieved at 1 mM of carbamylcholine or acetylcholine and at 0.1 microM of CCK-PZ or its octapeptide analog. Atropine displaced the dose-response curve for carbamylcholine to the right so that in the presence of 7 microM atropine a concentration of 1 mM carbamylcholine now gave an optimal rate of enzyme secretion. The ionophore A-23187 which bypasses the receptor and elicits enzyme secretion did not relieve the inhibition caused by supraoptimal concentrations of secretagogues, indicating that the inhibition occurs at the cellular rather than at the receptor level. Secretin had no effect on the inhibition of enzyme secretion by a high concentration of carbamylcholine, indicating that the inhibition was not caused by lack of water and electrolyte secretion. The energy-producing metabolism was not affected since the ATP level in the pancreatic slices was the same in the presence of either inhibitory or optimal concentrations of secretagogues. The inhibition of enzyme secretion was reversible since restoration of efficient enzyme secretion occurred after removal of carbamylcholine (1 mM) by washing, followed by addition of an optimal concentration of CCK-PZ octapeptide. Morphological studies revealed that the presence of inhibitory concentrations of secretagogues caused severe distortion of the lumen structure: disruption of the filamentous system surrounding the lumen, disappearance of microvilli, and production of distended evaginations of the luminal membrane containing cellular material. These changes eventually caused a reduction in the size of the lumen which becomes plugged with secretory material. It is suggested that these changes in the microtubular microfilamentous system could account for the inhibition of enzyme secretion.

Acetylcholine↗
PubMed1978

A transient release of potassium mediated by the action of substance P on rat parotid slices.

Substance P (a peptide of eleven amino acids) caused a Ca-dependent release of K+ from rat parotid slices. The response to substance P differed from the alpha-adrenergic and the cholinergic responses in that it was transient, of smaller extent, and was not inhibited by phentolamine and atropine. Substance P caused little, if any, amylase secretion. Successive additions of the peptide to the slice system maintained the effect of C+ release indicating that the transient response to a single addition of the peptide was due to inactivation of substance P and not due to a decline in the response of the tissue.

Amylases↗
PubMed1977

Mechanism of adenylate cyclase activation by cholera toxin: inhibition of GTP hydrolysis at the regulatory site.

Treatment of turkey erthrocyte membranes with cholera toxin caused an enhancement of the basal and catecholamine-stimulated adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] activities. Both of these activities required the presence of GTP. The toxin effect on the adenylate cyclase activity concided with an inhibition of the catecholamine-stimulated guanosinetriphosphatase activity. Inhibition of the guanosinetriphosphatase, as well as enhancement of the adenylate cyclase activity, showed the same dependence on cholera toxin concentrations, and the effect of the toxin on both activities was dependent on the presence of NAD. It is proposed that continuous GTP hydrolysis at the regulatory guanyl nucleotide site is an essential turn-off mechanism, terminating activation of the adenylate cyclase. Cholera toxin inhibits the turn-off guanosinetriphosphatase reaction and thereby causes activation of the adenylate cyclase. According to this mechanism GTP should activate the toxin-treated preparation of adenylate cyclase, as does the hydrolysis-resistant analog guanosine 5'-(beta,gamma-immino)triphosphate [Gpp(NH)p]. Indeed, the toxin-treated adenylate cyclase was maximally activated, in the presence of isoproternol, by either GTP or Gpp(NH)p, while adenylate cyclase not treated with toxin was stimulated by hormone plus GTP to only one-fifth of the activity achieved with hormone plus Gpp(NH)p. Furthermore, the toxin-treated adenylate cyclase activated by isoproterenol plus GTP remained active for and extended period (half-time of 3 min) upon subsequent addition of the beta-adrenergic blocker, propranolol. The native enzyme, however, was refractory to propranolol only if activated by Gpp(NH)p but not by GTP.

Adenylyl Cyclases↗
PubMed1977

The regulatory GTPase cycle of turkey erythrocyte adenylate cyclase.

It has recently been suggested that adenylate cyclase activity is controlled by a regulatory cycle consisting of two reactions: a hormone induced formation of the active adenylate cyclase-GTP complex, and a subsequent turn-off reaction in which hydrolysis of the bound nucleotide reverts the system to the inactive state. To test this model each of the two reactions was measured separately and their rate constants were used to estimate the steady state adenylate cyclase and GTPase activities. The first order rate constants were kon = 3 min-1 for the activation reaction and koff = 15 min-1 for the turn-off reaction. Substitution of these rate constants in the steady state equation of the regulatory cycle gave values of hormone stimulated adenylate cyclase and GTPase activities similar to those determined by direct measurements. Treatment of the adenylate cyclase with cholera toxin caused a decrease of 96% in the rate constant of the turn-off reaction. In this case too the activities calculated from the steady state equation were in good agreement with those determined directly.

Adenylyl Cyclases↗
PubMed1977

Catecholamine-induced release of [3H]-Gpp(NH)p from turkey erythrocyte adenylate cyclase.

Incubation of Gpp(NH)p-activated adenylate cyclase in the presence of isoproterenol caused the release of bound [3H]-Gpp(NH)p, and the decline of activity to the basal state. The isoproterenol-induced release of the nucleotide was proportional to the decrease in adenylate cyclase activity. Since there is a large excess of Gpp(NH)p binding sites in the membrane, the isoproterenol induced release of Gpp(NH)p, rather than binding of the nucleotide, was used to measure the amount of guanyl nucleotide binding sites coupled to the activated adenylate cyclase. This amount, 1.5-2.0 pmoles/mg membrane protein, is only approximately 1% of the total Gpp(NH)p binding sites, and is about equal to the number of beta-adrenergic receptors in the membrane. Chromatographic analysis revealed that Gpp(NH)p was released from the membrane as an intact molecule. The findings suggest that persistent activation of the adenylate cyclase is due to persistent binding of Gpp(NH)p to the regulatory site, and that this GTP analog is a better activator of the adenylate cyclase than GTP because of its resistance to hydrolysis.

Adenosine Triphosphate↗
PubMed1976

Catecholamine-stimulated GTPase activity in turkey erythrocyte membranes.

Determination of specific GTPase (EC 3.6.1.--) activity in turkey erythrocyte membranes was achieved using low concentration of GTP (0.25 muM), inhibition of nonspecific nucleoside triphosphatases by adenosine 5'(beta,gamma-imino-triphosphate (App(NH)p) and suppression of the transfer of gamma-32P from GTP to ADP with an ATP regeneration system. Under these conditions catacholamines caused a 30--70% increase in GTP hydrolysis. The stimulation of GTPase activity by catecholamines required the presence of Mg2+ or Mn2+. DIfferent batches of membranes revealed the following specific activities (pmol 32Pi/mg protein min): basal GTPase (determined in the absence of catecholamine), 6-- 11; catecholamine-stimulated TTPase, 3--7; and residual non-specific NTPase 3--5. The stimulation of GTPase activity by catecholamines fulfilled the stereospecific requirements of the beta-adrenergic receptor, and was inhibited by propranolol. The concentrations of DL-isoproterenol which half-maximally activated the GTPase and adenylate cyclase were 1 and 1.2 muM, respectively. The following findings indicate that the catecholamine-stimulated GTPase is independent of the catalytic production of cyclic AMP by the adenylate cyclase. Addition of cyclic AMP to the GTPase assay did not change the rate of GTP hydrolysis. Furthermore, treatment of the membrane with N-ethylmaleimide (MalNEt) at 0 degrees C which caused 98% inhibition of the adenylate cyclase, had no effect on the catecholamine-stimulated GTPase. The affinity and specificity for GTP in the GTPase reactions are similar to those previously reported for the stimulation of the adenylate cyclase. The apparent Km for GTP in the basal and the catecholamine-stimulated GTPase reaction was 0.1 muM. These GTPase activities were inhibited by ITP but not by CTP and UTP. It is proposed that a catecholamine-stimulated GTPase is a component of the turkey erythrocyte adenylate cyclase system.

Adenylyl Cyclases↗
PubMed1975

The functions of cyclic AMP and calcium as alternative second messengers in parotid gland and pancreas.

Biochemical and ultrastructural studies of rat parotid gland slices have led to the identification of alpha- and beta-adrenergic receptors and a cholinergic receptor, all operating within the same secretory cell. While cyclic AMP serves as the second messenger in the beta-adrenergic response of enzyme secretion, Ca++ serves as the second messenger in the alpha-adrenergic and in the cholinergic responses which both lead to K+ release and water secretion. Ca++ also serves as a second messenger for the muscarinic cholinergic receptor in rat pancreas slices in which it causes enzyme secretion. Analysis of this information leads to the conclusion that neither the neurotransmitter, nor the receptor, nor the second messenger are unique for a certain type of response. The latter seems to be dictated by a component of the specific response pathway which is affected by the second or a subsequent messenger. By having different neurotransmitters operate the same response and a single neurotransmitter operate different responses diversity of control is achieved.

Animals↗
PubMed1974

A calcium ionophore simulating the action of epinephrine on the alpha-adrenergic receptor.

Calcium acting through the ionophore A-23187 caused massive K(+) release from rat parotid slices. The ionophore thus simulates the action of epinephrine on the alpha-adrenergic receptor in this system. The alpha-adrenergic and cholinergic receptors are, however, not involved in the action of the ionophore since phetolamine and atropine had no effect on the ionophore-induced K(+) release. Amylase secretion, which is induced by epinephrine through the beta-adrenergic receptor but does not require Ca(++) in the medium, was only slightly increased by the ionophore in the presence of Ca(++). The K(+) released by Ca(++) through the ionophore was regained by the cells upon chelation of Ca(++) with ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid. Similar to the action of epinephrine on the alpha-adrenergic receptor, the K(+) release induced by the ionophore is specific for Ca(++), which cannot be substituted by 2.5 mM Sr(++), Mg(++), or Ba(++). The results suggest that epinephrine, like the ionophore, introduces Ca(++) into the cell to mediate K(+) release. It is proposed that the ionophore A-23187 may be an effective probe to test whether other Ca(++)-requiring hormones and neurotransmitters act in a similar fashion to produce their diverse physiological responses.

Acetates↗