Effect of ranakinin, a novel tachykinin, on cytosolic free calcium in frog adrenochromaffin cells.

Abstract

It has recently been shown that two novel tachykinins, ranakinin and [Leu3, Ile7]neurokinin A, are present in fibers innervating the frog adrenal gland, and it has been demonstrated that tachykinins stimulate corticosteroid secretion in vitro through activation of chromaffin cells. The purpose of the present study was to investigate the effect of ranakinin on cytosolic free calcium concentrations ([Ca2+]i) and to determine the source of calcium involved. Cultured adrenal cells were loaded with the fluorescent calcium indicator indo-1, and changes in [Ca2+]i were studied using dual emission wavelength microfluorimetry. Administration of a brief pulse of ranakinin (1 microM; 1 sec) in the vicinity of chromaffin cells caused an immediate and transient increase in [Ca2+]i. Repeated pulses of ranakinin resulted in a gradual decline in the [Ca2+]i response, suggesting the occurrence of a desensitization phenomenon. Preincubation of the cells with the calcium channel blockers nifedipine (10 microM) and omega-conotoxin (1 microM) did not alter the response of chromaffin cells to ranakinin. Chelation of extracellular calcium by EGTA (10 mM) caused a marked decrease in the basal [Ca2+]i, but did not suppress the ranakinin-induced [Ca2+]i increase. Conversely, incubation of the cells with thapsigargin (10 microM), an inhibitor of calcium adenosine triphosphatase activity, abolished the stimulatory effect of ranakinin, indicating that the increase in [Ca2+]i can be ascribed to mobilization of calcium from intracellular stores. Preincubation of adrenal cells with the phospholipase C antagonist U-73122 (1 microM; 18 min) or with pertussis toxin (10 microM; 18 h) totally blocked the ranakinin-induced [Ca2+]i rise. Taken together, these data indicate that in frog adrenochromaffin cells, ranakinin causes mobilization of calcium from intracellular stores. The effect of ranakinin is mediated through activation of a phospholipase C via a pertussis toxin-sensitive G protein.

Pituitary Adenylate Cyclase-activating Polypeptides Directly Stimulate Sympathetic Neuron Neuropeptide Y Release through PAC1 Receptor Isoform Activation of Specific Intracellular Signaling Pathways

“…Similar homologous phospholipase C inhibition with the aminosteroid 1-[6-((17␤-3-methoxyestra-1,3,5-(10)-triene-17-yl)amino)hexyl]-1H-pyrrole-2,5,-dione (U73122) (CalbiochemNovabiochem) decreased basal and PACAP27-or PACAP38-stimulated sympathetic neuron inositol phosphate production nearly 50% (p Ͻ 0.001; Fig. 8B, dark gray bars), in agreement with inhibitor effects reported in other systems (47)(48)(49)(50).…”

“…Inhibition of adenylyl cyclase did not affect sympathetic neuron PACAPmediated NPY release; by contrast, phosphatidylinositol signaling, presumably through inositol 1,4,5-trisphosphate-mediated intracellular calcium release, appeared to represent the predominant means of PACAP-stimulated sympathetic neurosecretion. Similarly, U73122 attenuated tachykinin-stimulated adrenal gland corticosteroid and aldosterone secretion and endothelin-1-and GnRH-induced luteinizing hormone release from gonadotropes (49,50). PACAP, primarily through PAC 1 (very short) receptors, also stimulates release of acetylcholine from cardiac ganglia parasympathetic neurons amplifying cardiac ganglia parasympathetic inhibition, but the intracellular signaling mechanisms coupled to these receptormediated events are unknown (40,71).…”

Activation of EndothelinA Receptors in Frog Adrenocortical Cells Stimulates Both Calcium Mobilization from Intracellular Stores and Calcium Influx through L-Type Calcium Channels

“…Conversely, in chromaffin cells, ET-1 (10 Ϫ6 m) has no effect on [Ca 2ϩ ] i (n ϭ 34), whereas the substance P analog ranakinin (10 Ϫ6 m) elicited a robust increase in [Ca 2ϩ ] i (Fig. 3, inset) as previously described (20). Application of graded concentrations of ET-1 (10 Ϫ12 to 10 Ϫ6 m) in the vicinity of adrenocortical cells produced a dose-dependent increase in [Ca 2ϩ ] i with an EC 50 of 1.7 ϫ 10 Ϫ9 m (Fig.…”

“…9C) 9B, inset), indicating that EGTA did not deplete the pool of intracellular Ca 2ϩ . In addition, we have previously shown that EGTA does not affect the response of frog adrenochromaffin cells to ranakinin (20).…”

The adrenergic stress response in fish: control of catecholamine storage and release

“…In the axillary bodies of the elasmobranch S. acanthias, there are also nerve fibers exhibiting substance P-like and galanin-like immunoreactivity [237]. Although the physiological significance of these observations remains speculative, in both mammals and amphibians there is increasing evidence that VIP, PACAP, and tachykinins (the family of peptides which includes substance P) may have the ability to stimulate and/or modulate catecholamine release under physiological conditions [54,112,145,146,165,167,278,286,302,306].…”