Ronald P. Rubin

Binding sites for inositol trisphosphate in the bovine adrenal cortex

Binding sites for inositol trisphosphate (IP3) have been identified in bovine adrenal cortex, employing [32P]IP3 prepared from human erythrocytes radiolabeled with [32P]ATP. IP3 was bound to adrenal microsomes with high affinity (Kd = 5 nM) and low capacity (186 fmol/mg protein). During kinetic studies, half-maximal binding was reached in less than one min at 4 degrees C, and dissociation was even more rapid with t1/2 of about 10 sec. [32P]IP2 showed no binding to the microsomal sites, which represent putative receptors at which IP3 acts to elevate intracellular calcium concentration during the actions of peptide hormones such as angiotensin II.

A guanine nucleotide-dependent regulatory protein couples substance P receptors to phospholipase C in rat parotid gland

Electrically permeabilized cells of rat parotid gland, prelabelled with [3H]-inositol, synthesized [3H]-inositol phosphates (IP3 and IP2) when stimulated with alpha 1-adrenergic, muscarinic-cholinergic, and substance P receptor-agonists. Non-hydrolyzable analogues of GTP (GTP gamma S and GppNHp) also stimulated [3H]-IP3 formation by permeabilized cells and they potentiated the stimulation by receptor-agonists. These effects of guanine nucleotides occurred only with GTP analogues and only in permeabilized cells indicating an intracellular site of action. NaF stimulated [3H]-IP3 accumulation, an effect that was not entirely attributable to the ability of F- to inhibit (1,4,5)IP3 degradation. These results suggest that a guanine nucleotide-dependent regulatory protein couples Ca2+-mobilizing receptors to phospholipase C in parotid gland.

Pertussis toxin inhibits chemotactic factor-induced phospholipase C stimulation and lysosomal enzyme secretion in rabbit neutrophils

Pertussis toxin suppressed [32P]polyphosphoinositide breakdown and lysosomal enzyme secretion induced by fMet‐Leu‐Phe in rabbit neutrophils. Likewise, fMet‐Leu‐Phe‐ or leukotriene B4‐evoked (3H]inositol trisphosphate accumulation was inhibited by the toxin. These findings, taken together with evidence that pertussis toxin specifically causes inactivation of the guanine nucleotide binding protein (Ni), suggests that guanine nucleotide binding proteins may mediate coupling between calcium‐mobilising receptors and phospholipase C‐mediated reactions in rabbit neutrophils.

NICOTINE-INDUCED STIMULATION OF STEROIDOGENESIS IN ADRENOCORTICAL CELLS OF THE CAT

1 The effect of nicotine on steroid production and release from trypsin‐dispersed cat adrenocortical cells was investigated. 2 Nicotine, like adrenocorticotrophin (ACTH), elicited a dose‐dependent increase in steroidogenesis, which depended upon the presence of calcium in the medium. 3 Augmented steroid production evoked by submaximal concentrations of ACTH, monobutyryl cyclic adenosine 3′,5′‐monophosphate (AMP), or prostaglandin E2 was further enhanced by steroidogenic concentrations of nicotine. 4 These results are discussed in relation to the possible mode of action of nicotine on cortical cells and to the potential consequences of smoking during stress.

Prostacyclin: A potent stimulator of adrenal steroidogenesis

The relative potencies of various prostaglandins were investigated in trypsin-dispersed cat adrenocortical cells. Prostacyclin proved to be the most potent steroidogenic prostaglandin, being 100-1000 times more potent than PGE2. This stimulant effect of prostacyclin was only partially dependent upon the presence of extracellular calcium and was associated with increased levels of cyclic AMP. These data suggest a possible role for prostacyclin in corticosteroidogenesis.

Indomethacin-induced alterations in corticosteroid and prostaglandin release by isolated adrenocortical cells of the cat.

1 The effects of purported prostaglandin synthesis inhibitors on steroid and prostaglandin (E and F) release from trypsin‐dispersed cat adrenocortical cells were investigated. 2 Low indomethacin concentrations potentiated adrenocorticotrophin (ACTH)‐evoked prostaglandin and steroid release, whereas higher concentrations depressed both responses to ACTH. The steroidogenic response to exogenous prostaglandin E2 was not markedly altered over a wide range of indomethacin concentrations. 3 Indomethacin enhanced basal steroid release but did not enhance basal prostaglandin E or F release. 4 5, 8, 11, 14‐Eicosatetraynoic acid (ETA) elicited a concentration‐dependent inhibition of ACTH‐induced steroid release, but had little effect on prostaglandin E2‐induced steroid release. A high concentration of ETA inhibited prostaglandin E and F release. 5 These data are discussed in relation to the concept that prostaglandins provide a critical link in ACTH‐induced corticosteroidogenesis.

Radioimmunoassay measurement of ACTH-facilitated PGE2 and PGF2α release from isolated cat adrenocortical cells

Prostaglandin (PG) biosynthesis by trypsin-dispersed cat adrenocortical cells was studied by radioimmunoassay (RIA). Parallel assays of incubation media using PGF2alpha and PGF1alpha antisera established that PGF2alpha is the primary PGF released by feline cortical cells. Following the reduction of PGE to PGF with sodium borohydride (NaBH4) these same two antisera were also used to identify PGE2 as the primary PGE released. RIA using a PGE antiserum confirmed the presence of PGE in the incubation medium. Steroidogenic concentrations of ACTH (50-250muU) enhanced PGE and PGF release, and indomethacin suppressed the ACTH-facilitated release. These studies provide additional evidence for ACTH-induced PG synthesis by feline cortical cells, and support the hypothesis that PGs play some role in the steroidogenic action of ACTH.

Secretagogues for lysosomal enzyme release as stimulants of arachidonyl phosphatidylinositol turnover in rabbit neutrophils

Summary [ 14 C]arachidonic acid incorporation into phosphatidylinositol from rabbit neutrophils pretreated with cytochalasin B was increased within 2 min by the synthetic peptide formyl-methionyl-leucyl-phenylalanine and the Ca 2+ ionophore A23187. A high concentration of the peptide elicited only a small increase in phosphatidylinositol turnover when [ 14 C]palmitic acid was employed as precursor. These data coincide with our earlier studies which identified a Ca 2+ -dependent increase in arachidonyl phosphatidylinositol turnover during adrenocorticotropin and A23187-induced activation of steroid production and release in adrenocortical cells. It is concluded that an increase in arachidonyl phosphatidylinositol turnover mediated by a Ca 2+ -dependent phospholipase A 2 may be a general mechanism for altering membrane function during secretory activity.

Inhibition of dibutyryl cyclic amp induced steroidogenesis in rat adrenocortical cells by the putative calcium antagonist TMB-8

A significant proportion of the steroidogenic response of isolated rat adrenocortical cells to dibutyryl cyclic AMP does not require extracellular calcium, and this component is profoundly depressed by low concentrations of the putative calcium antagonist, TMB-8. The inhibition is reversed by either the readdition of calcium or the calcium ionophore A23187. The steroidogenic response to pregnenolone, whose mode of action does not require calcium, was not depressed by TMB-8. Corticotropin (ACTH)-induced steroidogenesis, which requires extracellular calcium, was markedly depressed by TMB-8, although enhanced cyclic AMP formation is only slightly depressed by this drug. Adrenal cortical microsomes possess an ATP-dependent 45calcium (45Ca2+) uptake system which responded to EGTA with a rapid efflux of 45Ca2+; EGTA-induced calcium efflux from this microsomal fraction was markedly reduced by a concentration of TMB-8 that blocked dibutyryl cyclic AMP-evoked steroidogenesis. TMB-8 produced a smaller but significant reduction of EGTA-facilitated 45Ca2+ efflux from a mitochondrial-enriched fraction. We interpret these results to mean that TMB-8 blocks the steroidogenic effect of dibutyryl cyclic AMP by interfering with the mobilization of a cellular pool of calcium that is probably localized to the endoplasmic reticulum. The physiological implications of these findings in relation to the complex interactions between calcium and cyclic AMP in adrenal steroidogenesis are discussed.

On the role of cyclic AMP and cyclic GMP in steroid production by bovine cortical cells

The time course of corticotropin-induced steroidogenesis and changes in intracellular cyclic AMP and cyclic GMP levels were investigated in isolated bovine adrenocortical cells prepared by trypsin digestion. Corticotropin produced a peak rise in cyclic AMP during the first 5 min of stimulation and enhanced steroid production after 15 min. Corticotropin also caused a decrease in cortical cyclic GMP at 5 min; this decrease in cyclic GMP reverted to a 2-3 fold increase at 15-30 min which gradually subsided by 60 min. A steroidogenic concentration of prostaglandin E2 also produced an elevation in the levels of both nucleotides, but the rise in cyclic GMP preceded the rise in cyclic AMP. These results suggest that the relative amounts of cyclic AMP and cyclic GMP, rather than the absolute levels of cyclic AMP, may be a key factor in the regulation of steroidogenesis.