Philip D. Marley

High prealbumin and transferrin mRNA levels in the choroid plexus of rat brain

Expression of plasma protein genes in various parts of the rat brain was studied by hybridizing radioactive cDNA to RNA in cytoplasmic extracts. No mRNA could be detected in brain for the beta subunit of fibrinogen, major acute phase alpha 1-protein, alpha 1-acid glycoprotein and albumin. However, per g tissue, the choroid plexus contained at least 100 times larger amounts of prealbumin mRNA than the liver and about the same amount of transferrin mRNA as liver. No prealbumin mRNA was found in other areas of the brain. The results obtained suggest very active synthesis of prealbumin in choroid plexus, which would be an important link in the transport of thyroid hormones from the blood to the brain via the cerebrospinal fluid.

Histamine‐induced increases in cyclic AMP levels in bovine adrenal medullary cells

1 The effect of histamine on cellular cyclic AMP levels in cultured bovine adrenal medullary cells has been studied. 2 Histamine (0.3–30 μm) increased cyclic AMP levels transiently, with a maximal response after 5 min, a smaller response after 20 min, and no increase seen after 80 or 180 min. The EC50 at 5 min was approximately 2 μm. Histamine had no effect on cyclic AMP release from the cells over 5 min, but increased it after 90 min. 3 The cyclic AMP response to 5 μm histamine was reduced by 45% by 1 μm mepyramine and by almost 30% by 1 μm cimetidine, and was abolished by the combination of both antagonists. Cimetidine at 100 μm did not inhibit the response to histamine more than 1 μm cimetidine. The H3‐receptor antagonist, thioperamide (1 μm), had no effect on the response to histamine. 4 The H1‐receptor agonist, 2‐thiazolylethylamine (5–100 μm) and the H2‐receptor agonist, dimaprit (5–100 μm), each induced a cyclic AMP response, and gave more‐than‐additive responses when combined. The H3 agonist (R)α‐methylhistamine (100 μm) had no effect either on its own or in combination with either the H1 or the H2 agonist. The response to 100 μm 2‐thiazolylethylamine was unaffected by cimetidine (100 μm). 5 The cyclic AMP responses to 5μm histamine, 100 μm thiazolylethylamine and 100 μm dimaprit were each weakly enhanced in the presence of 1 mm 3‐isobutyl‐1‐methylxanthine. The response to dimaprit was enhanced more than 10 fold in the presence of 0.3 μm forskolin, while the responses to histamine and thiazolylethylamine were weakly enhanced. 6 The cyclic AMP response to 5 μm histamine was partially reduced in the absence of extracellular Ca2+, and the residual response was fully antagonized by 1 μm cimetidine and was unaffected by 1 μm mepyramine. In the absence of Ca2+, the cyclic AMP response to 100 μm thiazolylethylamine was abolished, while that to 100 μm dimaprit was unaffected. 7 Reincubation of 5 μm histamine solutions with a second set of chromaffin cells, following prior incubation with another set of cells, induced a cyclic AMP response in the fresh cells. This response was reduced by a combination of mepyramine and cimetidine to the same degree as the response to fresh 5 μm histamine solutions. 8 The results indicate that histamine increases cellular cyclic AMP levels in bovine chromaffin cells by three mechanisms: by acting on H1 receptors, by acting on H2 receptors, and by an interaction between H1 and H2 receptors. The H1 response does not require concomitant activation of H2 receptors, is fully dependent on extracellular Ca2+, does not depend on secreted chromaffin cell products, and is not due to reduced cyclic AMP degradation or export. The H2 cyclic AMP response is the first functional response reported for H2 receptors on chromaffin cells, is independent of Ca2+, is not due to reduced cyclic AMP export or degradation, and is likely to be mediated via a direct action through Gs. The role of these different mechanisms in the regulation of cyclic AMP‐dependent processes in chromaffin cells by histamine is under investigation.

Effects of opioid peptides and morphine on histamine‐induced catecholamine secretion from cultured, bovine adrenal chromaffin cells

1 The effect of opioid peptides and morphine on histamine‐induced catecholamine secretion has been studied in monolayer cultures of dispersed, bovine adrenal chromaffin cells. 2 Histamine‐induced a dose‐dependent secretion of both adrenaline and noradrenaline with a threshold dose of approximately 5 nM, an EC50 of 150 nM and maximal secretion at 10 μM. 3 Catecholamine secretion induced by 1 μM histamine was completely dependent on extracellular calcium, was inhibited in a dose‐dependent manner by mepyramine (1 nM‐1 μM), and was unaffected by cimetidine (10 μM) and hexamethonium (0.1 mM). 4 Dynorphin‐1–13 (1 nM‐20μM), metorphamide (0.1 nM‐10μM), morphine (1 nM − 0.1 mM) and diprenorphine (1 nM − 0.1 mM) each had no effect on adrenaline or noradrenaline secretion induced by 1 μM histamine. 5 The characteristics of histamine‐induced catecholamine secretion from bovine adrenal chromaffin cells were similar to those reported previously for cat and rat adrenal medulla being calcium‐dependent and mediated by H1 histamine‐receptors. The results with opioid peptides and morphine suggest that endogenous adrenal opioid peptides do not act on the opioid binding sites found on adrenal medullary chromaffin cells to modify their secretory response to histamine.

Chromogranin A: Secretion of Processed Products from the Stimulated Retrogradely Perfused Bovine Adrenal Gland

Chromogranin A (CGA) is a member of a family of highly acidic proteins co‐stored and co‐secreted with adrenaline and noradrenaline in the adrenal medulla. A number of biologically active fragments of CGA (CGAFs) have been characterized including a group of small N‐terminal fragments collectively named vasostatins due to their vascular inhibitory activity. In the present study, the release of CGAFs, including CGA N‐terminal fragments, from the isolated, retrogradely perfused bovine adrenal gland, has been studied under basal conditions and during nerve stimulation and perfusion with acetylcholine. The CGAFs were characterized by SDS‐PAGE followed by immunoblotting with antisera to specific sequences within the CGA molecule. Many different CGAFs were released during stimulation of the glands. Antisera to CGA1–40 and CGA44–76 detected a 7 kD protein whose release was increased during stimulation. This component co‐migrated with synthetic CGA1–76, was not immunoreactive to antisera to CGA79–113 or CGA124–143, and was seen whether or not the serine protease inhibitor aprotinin was present in the perfusion medium. The release of an ∼ 18 kD component, which stained with antisera to CGA1–40, CGA44–76 and CGA79–113, but not to chromostatin (CGA124–143), was also increased during stimulation. Components of 22 kD and larger were detected with antisera to chromostatin, but not with antisera to CGA1–40, CGA44–78 and CGA79–113. Two of these components of 22 to 24 kD were enhanced during nerve stimulation in the presence of aprotinin. The results indicate that processed Chromogranin A fragments are secreted from the bovine adrenal medulla during stimulation of chromaffin cells. The major fragments secreted appear to be the N‐terminal fragments of CGA, CGA1–76 and CGA1–113, which would arise as a result of processing of CGA at the first and second pairs of basic amino acids. A number of larger CGAFs, possibly containing the chromostatin sequence CGA124–143 at their N‐terminal, and components similar in size to intact CGA and to proteoglycan forms of CGA, are also secreted from the perfused bovine adrenal gland during stimulation.

Histamine promotes excitability in bovine adrenal chromaffin cells by inhibiting an M-current

The current study has investigated the electrophysiological responses evoked by histamine in bovine adrenal chromaffin cells using perforated‐patch techniques. Histamine caused a transient hyperpolarization followed by a sustained depolarization of 7.2 ± 1.4 mV associated with an increase in spontaneous action potential frequency. The hyperpolarization was abolished after depleting intracellular Ca2+ stores with thapsigargin (100 nm), and was reduced by 40 % with apamin (100 nm). Membrane resistance increased by about 60 % during the histamine‐induced depolarization suggesting inhibition of a K+ channel. An inward current relaxation, typical of an M‐current, was observed in response to negative voltage steps from a holding potential of −30 mV. This current reversed at −81.6 ± 1.8 mV and was abolished by the M‐channel inhibitor linopirdine (100 μm). During application of histamine, the amplitude of M‐currents recorded at a time corresponding with the sustained depolarization was reduced by 40 %. No inward current rectification was observed in the range −150 to −70 mV, and glibenclamide (10 μm) had no effect on either resting membrane potential or the response to histamine. The results show that an M‐current is present in bovine chromaffin cells and that this current is inhibited during sustained application of histamine, resulting in membrane depolarization and increased discharge of action potentials. These results demonstrate for the first time a possible mechanism coupling histamine receptors to activation of voltage‐operated Ca2+ channels in these cells.

Localization of angiotensin II binding sites in the bovine adrenal medulla using a labelled specific antagonist

Angiotensin II binding sites have been localized in sections of bovine adrenal glands and on living cultured bovine adrenal medullary cells using [125I]-[Sar1,Ile8]-angiotensin II and autoradiographic techniques. Binding sites were observed over both adrenaline and noradrenaline chromaffin cells. However, they were present in higher density over adrenaline cells, as determined by the distribution of phenylethanolamine N-methyltransferase mRNA by in situ hybridization histochemistry and of glyoxylic acid-induced fluorescence of noradrenaline. Binding sites were also observed in low density over nerve tracts within the bovine adrenal gland. Living cultured bovine adrenal medullary cells possessed angiotensin II binding sites. Not all cells were labelled. At least 73% of identified dispersed chromaffin cells in these cultures were labelled. Some chromaffin cells were not labelled with the ligand, and at least some non-chromaffin cells in the cultures did possess angiotensin II binding sites. The results provide direct anatomical support for the known ability of angiotensin II to elicit catecholamine secretion from perfused adrenal glands and from cultured adrenal chromaffin cells. They also suggest that some of the effects of angiotensin II on calcium fluxes and second messenger levels measured in cultured adrenal medullary cell preparations may be due to angiotensin II acting on non-chromaffin cells present in these cultures.

Evidence That Estrogen Suppresses Rho-Kinase Function in the Cerebral Circulation In Vivo

Background and Purpose— Premenopausal women are less susceptible to cardiovascular diseases than men or postmenopausal women. Such disease states are often associated with increased vascular RhoA/Rho-kinase activity and decreased activity of nitric oxide (NO). This study tested whether female gender is associated with lower Rho-kinase activity or higher NO activity in cerebral arteries in vivo and whether estrogen contributes to any such gender differences. Methods— Changes in basilar artery diameter were measured with the use of a cranial window preparation in anesthetized Sprague-Dawley rats. Some female rats were ovariectomized (OVX) and treated subcutaneously daily for 14 days with vehicle (dimethyl sulfoxide) or 17β-estradiol. Vascular expression of RhoA or Rho-kinase was assessed by Western blotting. Results— The Rho-kinase inhibitor Y-27632 was selectively ≈3-fold more potent as a cerebral vasodilator in males versus females. Expression of total RhoA or Rho-kinase did not differ between males and females. In OVX rats, vasodilator responses to Y-27632 resembled responses in males. Treatment of OVX rats with 17β-estradiol normalized the vasodilator effects of Y-27632 to be equivalent to responses in intact female controls. The NO synthase inhibitor N-nitro-l-arginine methyl ester caused ≈50% greater constriction of the basilar artery in females versus males, but responses in OVX rats treated with either vehicle or 17β-estradiol did not differ from those recorded in intact females. Conclusions— These data indicate that vascular Rho-kinase function is suppressed in females because of the effects of estrogen, whereas the higher NO activity in females is estrogen independent.

The role of sensory fibres in the rat splanchnic nerve in the regulation of adrenal medullary secretion during stress.

We have studied the involvement of sensory nerves containing substance P (SP) in the modulation of stress‐induced catecholamine (CA) secretion from the sympathetic nervous system and adrenal medulla. Adrenaline and noradrenaline (NA) levels were measured in blood samples withdrawn from the inferior vena cava (i.v.c.) at 5 or 15 min intervals for periods of up to 60 min, in adult rats during stress induced by insulin or cold. Insulin stress caused a biphasic elevation of plasma CA. Previous studies from our laboratory have shown that the first phase lasting 30 min is neurogenic, and the second phase from 30 to 60 min is non‐neurogenic in mechanism. In control adult rats (with normal levels of SP in their splanchnic nerve), insulin stress caused a slow and progressive secretion of adrenaline into the circulation for the first 30 min (neurogenic phase). In the period 30‐60 min (non‐neurogenic phase) plasma adrenaline and NA levels rose at a much higher rate. In capsaicin‐pre‐treated rats (in which SP levels in the splanchnic nerve were depleted by 68%) insulin stress produced a steady increase in plasma adrenaline levels for up to 5 min similar to that in insulin‐stressed control animals; however, by 10 min the plasma adrenaline levels had fallen to basal and remained low up to 30 min. From 30 to 60 min, plasma adrenaline and NA levels rose steeply as seen with control animals. We conclude that capsaicin pre‐treatment affected the neurogenic phase but did not affect the non‐neurogenic phase. Cold stress increased the plasma adrenaline levels by a neurogenic mechanism over 30 min in control rats. In contrast, in capsaicin‐pre‐treated, cold‐stressed rats, plasma adrenaline did not increase significantly. Plasma NA levels were also significantly lowered in capsaicin‐pre‐treated, cold‐stressed rats during the neurogenic phase but NA increases were not dependent on an intact adrenal innervation. The results using both insulin stress and cold stress suggest that capsaicin‐sensitive (sensory) nerve fibres in the adrenal medulla and in sympathetic ganglia are capable of modifying the secretory responses of these tissues to stress. Results from our previous in vitro work are compatible with the view that SP may be the neuromodulator released from such sensory nerves to produce these effects. This suggests that the previously reported ability of SP to modulate nicotinic receptor function in vitro by either inhibiting the nicotinic response or protecting against nicotinic desensitization may be more than a mere pharmacological curiosity.(ABSTRACT TRUNCATED AT 400 WORDS)

Differences between the mechanisms of adrenaline and noradrenaline secretion from isolated, bovine, adrenal chromaffin cells

Cultured, bovine, adrenal medullary chromaffin cells have been used to study the secretion of adrenaline (A) and noradrenaline (NA) in response to two consecutive stimulation periods. Cells were first exposed to nicotine, then rapidly washed before being exposed to elevated K+ levels. Both A and NA cell types secreted their catecholamine in response to nicotine; however, NA cells secreted 3 times more of their catecholamine stores as did A cells. The NA cells were desensitized to stimulation by K+ if they had previously been exposed to nicotine. A cells, on the other hand, were up to twice as responsive to K+ stimulation after having been stimulated with nicotine, in spite of substantial depletion of their catecholamine stores. This sensitization of A cells to K+ was neither due to carryover of nicotine from the first stimulation period, nor to accumulation of calcium in the cells during the nicotinic stimulation. The results suggest that stimulus-secretion coupling or the exocytotic machinery in A and NA chromaffin cells is different. These differences may contribute to the ratio A:NA secreted from the adrenal medulla during stress.

Nerves containing nitric oxide synthase and their possible function in the control of catecholamine secretion in the bovine adrenal medulla

NADPH-diaphorase reactivity and neuronal nitric oxide synthase (nNOS) immunostaining have been localised in sections of bovine adrenal glands. Both were present in nerve fibres and terminals in the subcapsular region and running between zona glomerulosa cells, amongst the medullary chromaffin cells, between large ganglion cells in rare encapsulated medullary ganglia and in large nerve bundles running through the cortex. Occasional isolated fibres were stained in deeper cortical layers. Both NADPH-diaphorase reactivity and nNOS immunoreactivity were present in a population of ganglion cells located individually or in small groups at the medullary-cortical boundary. NADPH-diaphorase reactivity was also found in all cortical cells (zona glomerulosa cells being more densely stained than other cortical cells) and in large fibrous structures in large nerve bundles (tentatively identified as glial cells): these structures were not stained with antisera to nNOS. Chromaffin cells were not stained with either technique. The possible role of neurally-released nitric oxide in the regulation of nerve-induced catecholamine secretion from chromaffin cells was investigated in isolated, perfused, bovine adrenal glands. The secretion of both adrenaline and noradrenaline in response to field stimulation of adrenal nerves at either 2 Hz or 10 Hz was unaffected by the presence of N omega-nitro-L-arginine (30 microM), sodium nitroprusside (10 microM) or L-arginine (100 microM) in the perfusing solution. It is concluded that, although nitric oxide may be generated and released from adrenal medullary nerves innervating chromaffin cells, it does not play a direct role in the acute regulation of adrenal catecholamine secretion.