Elizabeth T. McNeal

Accumulations of Cyclic AMP in Adenine‐Labeled Cell‐free Preparations from Guinea Pig Cerebral Cortex: Role of α‐Adrenergic and H1‐Histaminergic Receptors

Norepinephrine, histamine, adenosine, glutamate, and depolarizing agents elicit accumulations of radioactive cyclic AMP from adenine‐labeled nucleotides in particulate fractions from Krebs‐Ringer homogenates of guinea pig cerebral cortex. The particulate fractions contain sac‐like entities, which apparently are associated with a significant portion of the tnembranal adenylate cyclase. Particulate fractions from sucrose homogenates are a less effective source of such responsive entities. Activation of the adenine‐labeled cyclic AMP‐generating systems by norepinephrine is by means of α‐adrenergic receptors, while activation by histamine is through H1‐ and H2‐histaminergic receptors. Adenosine responses are potentiated by the amines and are antagonized by alkylxanthines. Glutamate and depolarizing agents appear to elicit accumulations of cyclic AMP via „release” of endogenous adenosine. It is proposed, based on the virtual absence of an α‐adrenergic or H1‐histaminergic response in the presence of a combination of potent adenosine and H2‐histaminergic antagonists, that α‐adrenergic and H1‐histaminergic receptor mechanisms do not activate adenylate cyclase directly in brain slices or Krebs‐Ringer particulate fractions, but merely facilitate activation by β‐adrenergic, H2‐histaminergic, or adenosine receptors.

Membrane Potentials in Cell-free Preparations from Guinea Pig Cerebral Cortex: Effect of Depolarizing Agents and Cyclic Nucleotides

The distribution of [3H]triphenylmethylphosphonium ion between the medium and vesicular entities was examined in a cell‐free, particulate preparation from guinea pig cerebral cortex. This distribution followed the Nernst relationship with regard to the external potassium ion concentration and, in physiological media, indicated the maintenance of a mean trans‐membrane potential ranging from –58 to –78 mV. The neurotoxins batrachotoxin, veratridine, and grayanotoxin I, partially depolarized the preparation. Tetrodotoxin blocked the depolarization by batrachotoxin, veratridine, and grayanotoxin I. The depolarization by these neurotoxins was potentiated by the presence of anemone toxin II and presumably reflected the response of vesicular components of neuronal origin. An additional potassium‐sensitive depolarization probably represented the response of vesicular components of glial origin with an apparent transmembrane potential of –8 to –35 mV. No correlation could be demonstrated between changes in transmembrane potential and stimulation of cyclic AMP generation by a variety of agents in this preparation.

Cyclic AMP-generating systems in cell-free preparations from guinea pig cerebral cortex: loss of adenosine and amine responsiveness due to low levels of endogenous adenosine.

The accumulations of radioactive cyclic AMP elicited by adenosine, norepinephrine, and histamine in adenine‐labeled vesicular entities of a particulate fraction from guinea pig cerebral cortex are greatly reduced as a result of prolonged preincubation. The presence of adenosine deaminase during preincubations largely prevents the loss of adenosine, norepinephrine and histamine responses. Adenosine deaminase was inactivated by deoxycoformycin prior to stimulation of cyclic AMP accumulation by adenosine or amines. If adenosine deaminase is not inactivated, responses to norepinephrine are not significant and histamine responses are reduced by 50%. Adenosine deaminase cannot restore responsiveness of the cyclic AMP‐generating systems. It is proposed that, in participate fractions of guinea pig cerebral cortex, low levels of adenosine cause a slow loss of receptors and/or of coupling of receptors to cyclic AMP‐generating systems.

Cardiotonic activities of pumiliotoxin B, pyrethroids and a phorbol ester and their relationships with phosphatidylinositol turnover

The cardiotonic activities of pumiliotoxins, pyrethroids and sodium and calcium channel activators were assessed in vitro with spontaneously beating guinea pig atria. The ability of these compounds to stimulate phosphoinositide turnover was assessed in guinea pig cerebral cortical synaptoneurosomes. The activity of pumiliotoxins for both cardiotonic activity and phosphoinositide breakdown was strongly dependent on the structure and configuration of the side chain and there was a correlation between structure and activity in the two systems. Pyrethroids that had cardiotonic activity also induced phosphoinositide breakdown. Other sodium channel and calcium channel activators that induced phosphoinositide breakdown were also cardiotonic. It is suggested that phosphoinositide breakdown leading to inositol phosphates and diacylglycerols may represent a mechanism underlying the cardiotonic effects of certain agents. A phorbol ester, phorbol 12-myristate 13-acetate, that mimics the activation of protein kinase C elicited by diacylglycerols, had cardiotonic activity.

Voltage-dependent sodium channels in synaptoneurosomes: studies with22Na+ influx and [3H]saxitoxin and [3H]batrachotoxinin-A 20-α-benzoate binding. Effects of proparacaine isothiocyanate

22Na+ influx and binding of [3H]saxitoxin ([3H]STX) and [3H]batrachotoxin-A 20-alpha-benzoate ([3H]BTX-B) were studied in guinea pig cerebral synaptoneurosomes. STX and tetrodotoxin (TTX) completely blocked the stimulation of sodium influx induced by 1 microM BTX. The IC50 values for STX and TTX closely matched the Ki values for inhibition of [3H]STX binding, suggesting that the sites labelled by [3H]STX are associated with a population of BTX-sensitive channels. BTX induced a dose-dependent stimulation of sodium influx in synaptoneurosomes (EC50 280 nM). The potency of BTX for stimulation of sodium influx was increased (EC50 24 nM) in the presence of 0.6 microgram/ml scorpion venom without any change in maximal influx. In contrast, specific binding of [3H]BTX-B to synaptoneurosomes was minimal in the absence of scorpion venom, but it was increased several fold in the presence of 60 micrograms/ml scorpion venom. With proparacaine isothiocyanate (PROPRIT), an irreversible local anesthetic, the inhibition of [3H]BTX-B binding by PROPRIT did not occur in parallel with an inhibition of sodium influx induced by BTX. Preincubation of synaptoneurosomes with 10 microM PROPRIT for 10 min resulted in approximately 70% inhibition of [3H]BTX-B binding in the presence of scorpion venom. Such preincubation did not alter BTX-induced sodium uptake in synaptoneurosomes. Preincubations of synaptoneurosomes with 100 microM PROPRIT for 10 min completely inhibited [3H]BTX-B binding, and under these conditions BTX-induced sodium influx was reduced only by 50%. The results indicate that virtual elimination of binding sites labeled by [3H]BTX-B in the presence of scorpion venom by PROPRIT has little effect on sodium influx induced by BTX.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of ring fluorination on the adrenergic properties of phenylephrine

The adrenergic properties of 2-, 4- and 6-fluorophenylephrine (2-FPE, 4-FPE, 6-FPE) were compared to those of phenylephrine (PE). The order of affinities of these compounds for alpha 1-adrenoceptors as determined by displacement of [3H]prazosin and [3H]WB-4101 binding to brain membranes was the same as the order of potencies for eliciting two alpha 1-adrenergic metabolic responses in guinea-pig cerebral cortical synaptoneurosomes, namely the stimulation of phosphatidylinositol turnover and the potentiation of 2-chloroadenosine-induced accumulation of cyclic AMP. In all cases the order was 6-FPE greater than PE greater than 4-FPE greater than 2-FPE. The order of affinities for alpha 2-adrenoceptors as determined by displacement of binding of [3H]clonidine to brain membrane was 6-FPE greater than PE greater than or equal to 4-FPE = 2-FPE. In contrast, the order of potencies for inhibition of forskolin-stimulated adenylate cyclase activity in human platelet membranes via an alpha 2-adrenoceptor was 6-FPE approximately equal to PE greater than 4-FPE much greater than 2-FPE. The FPEs and PE were partial agonists compared to epinephrine in human platelets. The affinities of these compounds for beta-adrenoceptors as determined by displacement of binding of [3H]dihydroalprenolol to brain membranes are 2-FPE greater than PE greater than or equal to 4-FPE much greater than 6-FPE. The FPEs and PE had positive chronotropic and inotropic effects in isolated guinea-pig atria apparently through the activation of a beta-adrenoceptor, since pindolol blocked the response while prazosin did not. 6-FPE appeared less active than the other PEs in atria. In fat cell membranes, 2-FPE was more potent than PE in stimulating adenylate cyclase via a beta-adrenoceptor, while 4-FPE and 6-FPE were inactive. Both, 2-FPE and PE were partial agonists in fat cells compared to isoproterenol. Of the three FPEs, 6-FPE represents a more potent and more selective agonist for alpha-adrenoceptors compared to beta-adrenoceptors than PE, while 4-FPE and, in particular, 2-FPE are less potent and selective as alpha-agonists.