Funding of evidence included within public comments submitted to inform Medicare national coverage determinations.

The Centers for Medicare & Medicaid Services (CMS) relies on public comments submitted in response to proposed national coverage determinations to assist the agency in determining the coverage of items and services for Medicare beneficiaries. In a cross-sectional study, we characterized the cited evidence and what funding supported the cited evidence submitted in public comments to CMS for all therapeutic medical device national coverage determinations finalized between June 2019 and June 2022. Of 681 public comments, 159 (23%) cited at least 1 identifiable published scientific journal article. Within these 159 public comments, 198 unique articles were cited, 170 (86%) of which included funding statements or author disclosures. Among these, 96 (56%) disclosed funding from manufacturers that would benefit from Medicare coverage and/or were written by author(s) who received funding from these manufacturers. In summary, most public commenters for national coverage determinations did not cite published scientific journal articles to support their positions. Among those who did, more than half of articles were directly funded by manufacturers that would benefit from coverage. Greater funding of independent, non-industry-supported research may help provide unbiased evaluations of benefits and harms to support Medicare coverage decisions.

Different contributions of voltage-sensitive Ca2+ channels to histamine-induced catecholamine release and tyrosine hydroxylase activation in bovine adrenal chromaffin cells.

Histamine stimulates catecholamine release and tyrosine hydroxylase activity in a Ca(2+)-dependent manner in bovine adrenal chromaffin cells. The role of voltage-sensitive Ca2+ channels in these two responses has been investigated. Using an EC50 concentration of histamine, 1 microM, catecholamine release was enhanced by (+/-)BayK8644, and partially inhibited by nitrendipine and omega-agatoxin IVA, blockers of L- and P/Q-type Ca2+ channels. omega-Conotoxin GVIA gave small and variable inhibitory effects. With a maximal histamine concentration, 10 microM, similar results were obtained except that now omega-conotoxin GVIA reliably reduced release. In contrast, neither (+/-)BayK8644 nor any of the individual Ca2+ channel antagonists had any significant effect on tyrosine hydroxylase (TOH) activation induced by either an EC50 or a maximal concentration of histamine. When high concentrations of nitrendipine, omega-conotoxin GVIA and omega-agatoxin IVA were combined with omega-conotoxin MVIIC (a non-selective blocker of N, P and Q channels) to block voltage-sensitive Ca2+ channels in these cells, release induced by K+ depolarization was completely blocked. Release caused by histamine, however, was substantially reduced but not abolished. The combination of antagonists also only partially inhibited TOH activation by histamine. The results show that the G protein-coupled receptor agonist histamine activates several different types of voltage-sensitive Ca2+ channels in chromaffin cells to mediate its cellular effects. Histamine may also activate additional pathways for Ca2+ entry. The results also suggest that the manner by which Ca2+ controls release and TOH activation once it has entered chromaffin cells through these channels are different.

Elevation in plasma catecholamines in response to insulin stress is under both neuronal and nonneuronal control.

We have examined the contribution of neurogenic and nonneurogenic influences to the secretion of adrenal catecholamines (CA) in adult rats after iv administration of insulin. Plasma CA levels were measured in control rats and in rats after surgical or pharmacological adrenal denervation or adrenalectomy. Insulin-induced CA secretion was biphasic and proportional to the insulin dose used. The first phase was neurogenic in origin and produced a moderate increase in plasma adrenaline (A) levels, with little or no change in noradrenaline (NA) levels. This neurogenic increase in plasma A was reduced by partial denervation and abolished by surgical complete adrenal denervation, adrenalectomy, or administration of hexamethonium and atropine. The second phase occurred later and produced a dramatic increase in both plasma A and NA. This phase was initiated when the plasma glucose level fell below 75 mg/100 ml. This late release of A and NA was not altered by surgical or pharmacological adrenal denervation, showing that it was nonneuronal in origin. However, both the early and late phases were abolished by adrenalectomy, showing that adrenal secretion of CA was the origin of the increased plasma levels of NA and A. The late rise in plasma CA was also abolished by iv administration of glucose. These data suggest that the mechanism responsible for the nonneurogenic secretion of adrenal CA in response to insulin stress was sensitive to the level of hypoglycemia.

Localization and actions of cholecystokinin in the rat pituitary neurointermediate lobe.

Rat pituitary neural lobe contained high concentrations of cholecystokinin-like immunoreactivity (CCK-LI). Section of the pituitary stalk resulted in loss of CCK-LI, and both lactation and replacement of drinking water with 2% saline resulted in marked depletion of CCK-LI. Rats with congenital diabetes insipidus (Brattleboro strain) had a 73% reduction in CCK-LI below the levels of hooded Long-Evans controls, where as levels in the brain were unchanged. Release of CCK-LI, labeled dopamine, and gamma-amino butyric acid in response to potassium depolarization was studied. There was a low fractional release of CCK-LI. Addition of sulfated CCK-8 (CCK-8s) to the medium enhanced the calcium-dependent potassium-stimulated release of dopamine, but basal release was unaffected. gamma-Amino butyric acid release was only poorly calcium dependent and not effected by extracellular CCK-8s. Vasopressin and oxytocin release were stimulated by electrical stimulation of the pituitary stalk, and were unaffected by the addition of CCK-8s to the medium. In vivo, however, the injection of 5 micrograms CCK-8s into the third ventricle resulted in increased plasma vasopressin concentrations.

Presence of angiotensin converting enzyme in the adventitia of large blood vessels.

BACKGROUND: Angiotensin converting enzyme (ACE) is present in the endothelial cells of all vascular beds. There are, however, many reports of converting enzyme activity in blood vessels not associated with the endothelium. METHODS: ACE was localized in large blood vessels of a number of mammals by in vitro autoradiography using the radioligand 125I-351A. To characterize this binding further, immunohistochemistry was performed on rabbit aorta using polyclonal antisera raised to two different preparations of rabbit lung ACE. RESULTS: In all of the blood vessels studied, which included the rabbit pulmonary artery, rabbit, dog and sheep aorta, human internal mammary artery and human saphenous vein, high levels of radioligand binding were found in endothelial cells, as expected. In addition, a very high density of punctate binding was observed interspersed between diffuse moderate labelling in the adventitia. Immunoreactivity was confined to the endothelium of both the intima and the vasa vasorum of the adventitia. The immunostaining correlated well with the autoradiography. The ACE inhibitors lisinopril and perindoprilat displayed similar high affinities in competing for the binding of 125I-351A to the endothelium and adventitia of the sheep aorta, suggesting that at these two sites the radioligand was binding to ACE. CONCLUSIONS: We find that ACE in the adventitia of large blood vessels is confined to the vaso vasorum. The results of this study help to explain the findings of many studies that ACE activity persists in endothelium-denuded blood vessels and also reveals a source of ACE distant from the luminal endothelial surface.

The distribution of opioid binding subtypes in the bovine adrenal medulla.

Autoradiography has been used to examine the distribution of opioid binding subtypes in the bovine adrenal gland. Specific opioid binding sites were restricted to the adrenal medulla. Kappa sites, labelled with [3H]bremazocine (in the presence of excess unlabelled mu and delta ligands), were highly concentrated over nerve tracts. These nerve tract associated binding sites were sensitive to competition by the endogenous opioid, dynorphin (1-13). Specific [3H]bremazocine binding sites were also found over the adrenal medullary chromaffin tissue. These binding sites were concentrated over the peripheral, adrenaline-containing region of the medulla and were sensitive to competition by diprenorphine but not dynorphin (1-13). Delta opioid sites, labelled with [3H][D-Ala2,D-Leu5] enkephalin (in the presence of excess unlabelled mu ligand) were selectively localized to the central, noradrenaline-containing region of the adrenal medulla. Mu opioid sites, labelled with [3H][D-Ala2, NMePhe4,Gly-ol5]enkephalin, were low in number and distributed throughout the adrenal medulla. These studies demonstrate that mu, delta and two distinct kappa opioid binding sites are differently distributed within the bovine adrenal medulla and suggest possible new sites of action for the adrenal medullary opioid peptides.

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.

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

The effect of opioid peptides and morphine on histamine-induced catecholamine secretion has been studied in monolayer cultures of dispersed, bovine adrenal chromaffin cells. 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 microM. Catecholamine secretion induced by 1 microM histamine was completely dependent on extracellular calcium, was inhibited in a dose-dependent manner by mepyramine (1 nM-1 microM), and was unaffected by cimetidine (10 microM) and hexamethonium (0.1 mM). Dynorphin-1-13 (1 nM-20 microM), metorphamide (0.1 nM-10 microM), 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 microM histamine. 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.

Inhibition of nicotinic responses of bovine adrenal chromaffin cells by the protein kinase C inhibitor, Ro 31-8220.

1. The effects of the protein kinase C inhibitor, Ro 31-8220, on the responses of cultured bovine adrenal chromaffin cells to nicotine, phorbol 12, 13-dibutyrate (PDBu) and K+ have been investigated. 2. Tyrosine hydroxylase activity was measured in situ in intact cells by measuring 14CO2 evolved following the hydroxylation and rapid decarboxylation of [14C]-tyrosine offered to the cells. Secretion of endogenous adrenaline and noradrenaline was measured by use of h.p.l.c. with electrochemical detection. Cyclic AMP levels were measured in cell extracts by RIA. 3. Ro 31-8220 produced a concentration-dependent inhibition of 300 nM PDBu-stimulated tyrosine hydroxylase activity with an IC50 of < 2 microM and complete inhibition at 10 microM. It had no effect on the responses to forskolin. 4. Ro 31-8220 produced a concentration-dependent inhibition of 5 microM nicotine-stimulated tyrosine hydroxylase activity, adrenaline and noradrenaline secretion and cellular cyclic AMP levels, with an IC50 of about 3 microM and complete inhibition by 10 microM. At concentrations up to 10 microM, Ro 31-8220 had little or no effect on the corresponding responses to 50 mm K+. 5. A structural analogue of Ro 31-8220, bisindolylmaleimide V, that lacks activity as a protein kinase C inhibitor, had no effect up to 10 microM on PDBu-stimulated tyrosine hydroxylase activity or on nicotine-stimulated cyclic AMP levels or noradrenaline secretion and only marginal inhibitory effects on nicotine-stimulated tyrosine hydroxylase activity and adrenaline secretion. 6. A structurally related protein kinase C inhibitor, bisindolylmaleimide I, inhibited PDBu-stimulated tyrosine hydroxylase activity with an IC50 of < 1 microM and complete inhibition by 3 microM, but had essentially no effect on nicotine stimulated tyrosine hydroxylase activity or catecholamine secretion. 7. The results suggest that Ro 31-8220 is not only a protein kinase C inhibitor but is also a potent inhibitor of nicotinic receptor responses in adrenal chromaffin cells by a mechanism unrelated to protein kinase C inhibition. The results are consistent with Ro 31-8220 being a nicotinic receptor antagonist.

Effects of N- and L-type calcium channel antagonists and (+/-)-Bay K8644 on nerve-induced catecholamine secretion from bovine perfused adrenal glands.

1. The effects of N- and L-type calcium channel antagonists and (+/-)-Bay K8644 on catecholamine release from chromaffin cells and acetylcholine release from splanchnic nerve terminals was investigated in bovine perfused adrenal glands. 2. Adrenal glands were perfused retrogradely and preloaded with [3H]-choline. Subsequent efflux of 3H-labelled compounds was taken as an index of acetylcholine release from the splanchnic nerve terminals. Noradrenaline and adrenaline release from the glands was measured by h.p.l.c. with electrochemical detection. 3. A maximally effective frequency of field stimulation of the adrenal nerves, 10 Hz, induced release of catecholamines and 3H-labelled compounds. Tetrodotoxin (1 microM) abolished release of both catecholamines and 3H-labelled compounds. A combination of mecamylamine (5 microM) and atropine (1 microM) inhibited nerve-induced catecholamine release by about 75% but did not inhibit release of 3H-labelled compounds. Reducing the concentration of extracellular calcium 5 fold to 0.5 mM inhibited nerve-induced catecholamine release by 80% and release of 3H-labelled compounds by 50%. 4. (+/-)-Bay K8644 (1 microM), nitrendipine (1 microM), omega-conotoxin-GVIA (10 nM) and the combination of nitrendipine and omega-conotoxin-GVIA each had no effect on nerve-induced release of 3H-labelled compounds. 5. (+/-)-Bay K8644 (1 microM) potentiated nerve-induced catecholamine release by 75%. Nitrendipine (1 microM) reduced release by 20% but this did not reach statistical significance, omega-Conotoxin-GVIA (10 nM) reduced nerve-induced catecholamine release by 75%, while the combination of omega-conotoxin-GVIA and nitrendipine reduced release to the same extent as omega-conotoxin-GVIA alone. 6. Exogenous acetylcholine perfusion through the glands produced a concentration-dependent increase in catecholamine release. The maximally effective concentration of acetylcholine for catecholamine release was > or = 300 microM, while 30 microM acetylcholine gave comparable catecholamine release to that obtained with 10 Hz field stimulation. 7. (+/-)-Bay K8644 (1 microM), nitrendipine (1 microM) and omega-conotoxin-GVIA (10 nM) each had no significant effect on catecholamine release evoked by perfusion of the gland with either a near maximally effective concentration of acetylcholine, 100 microM, or with the lower concentration of 30 microM. 8. The results show that the omega-conotoxin-GVIA-sensitive N-type voltage-sensitive calcium channels located on the chromaffin cells are largely responsible for catecholamine release induced by nerve stimulation in bovine adrenal glands. In contrast, N-type calcium channels are not involved in catecholamine release induced by exogenous acetylcholine. L-type voltage sensitive calcium channels do not play a major role in nerve-induced or exogenously applied acetylcholine-induced catecholamine release. However, the L-type calcium channels do have the potential to augment powerfully nerve-induced catecholamine release. N- and L-type calcium channels do not play a major role in the presynaptic release of acetylcholine.

Cholinoceptor regulation of cyclic AMP levels in bovine adrenal medullary cells.

1. The regulation of adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels by cholinoceptors has been studied in cultured bovine adrenal medullary cells. 2. Acetylcholine (100 microM), nicotine (10 microM) and dimethylphenylpiperazinium (20 microM) each increased cellular cyclic AMP levels 2 to 4 fold over 5 min in the absence of phosphodiesterase inhibitors. The muscarinic agonist acetyl-beta-methylcholine (100 microM) had no effect either on its own or on the response to nicotine. The responses to acetylcholine and nicotine were unaffected by atropine (1 microM) but were abolished by mecamylamine (5 microM). 3. Cellular cyclic AMP increased transiently during continuous exposure to nicotine (1-20 microM), with the largest response seen after 5 min, a smaller response after 20 min, and no change in cyclic AMP levels seen after 90 or 180 min. The maximal response after 5 min stimulation was seen with 5-10 microM nicotine and the EC50 was about 2 microM. In contrast, extracellular cyclic AMP levels did not change after 5 or 20 min stimulation with nicotine, but increased slightly after 90 min and further after 180 min. 4. The cellular cyclic AMP response to nicotine (10 microM) was unchanged or weakly enhanced in the presence of the unselective phosphodiesterase inhibitor, isobutylmethylxanthine, and was unchanged in the presence of rolipram. Nicotine did not interact synergistically with low concentrations of forskolin. The response was however completely abolished in the absence of extracellular Ca2+.

Protein kinase A and nicotinic activation of bovine adrenal tyrosine hydroxylase.

1. Stimulation of nicotinic cholinoceptors on bovine chromaffin cells increases phosphorylation of three serine residues in tyrosine hydroxylase (TOH) and activates TOH. One of the serines is a target for protein kinase A phosphorylation, and phosphorylation of this serine is adequate alone to cause TOH activation. The role of protein kinase A in nicotinic activation of TOH was therefore investigated. 2. TOH activity was studied in situ in intact, cultured, bovine adrenal chromaffin cells, by measuring 14CO2 evolved following the hydroxylation and rapid decarboxylation of [14C]-tyrosine offered to the cells. 3. Nicotine (5 microM), forskolin (1 microM) and 8-bromo-cyclic AMP (8-Br-cyclic AMP, 1 mM) each increased TOH activity by up to 200% over 10 min. The effect of nicotine was completely abolished by removal of extracellular Ca2+. 4. TOH activation by all three drugs was blocked by H89 (3-20 microM), which inhibits protein kinase A by competing for the ATP binding site on the kinase. Adenosine 3':5'-cyclic monophosphorothioate Rp-diastereomer (Rp-cAMPS) (1 mM), an inhibitor of protein kinase A that competes with cyclic AMP for the regulatory subunit of the kinase, abolished the activation of TOH by nicotine, and reduced that by forskolin and 8-Br-cyclic AMP. Both H89 and Rp-cAMPS inhibited basal TOH activity by 50-80%. 5. A structural analogue of H89, H85 (3-20 microM), which lacks activity as a protein kinase A inhibitor, did not inhibit either the activation of TOH by nicotine (5 microM) or basal TOH activity. Neither sodium nitroprusside (0.3-1O microM) nor 8-Br-cyclic GMP (1 mM) increased TOH activity.6. In digitonin-permeabilized chromaffin cells, forskolin (3 microM), cyclic AMP (10 microM) and Ca2+ (approx.2 micro M free Ca2+) each increased TOH activity. The response to all three drugs was blocked by H89(10 microM), which also reduced basal TOH activity in the permeabilized cells.7. Maximal activation of TOH by forskolin was achieved with 10 micro M forskolin. This concentration was less than the EC50 for forskolin-induced cyclic AMP accumulation in these cells. The activations of TOH by forskolin (1O microM) and nicotine (5 microM) were additive.8. The results indicate that both basal TOH activity and nicotinic activation of TOH in bovine chromaffin cells require protein kinase A activity. However, it is unlikely that nicotinic activation of TOH is directly mediated by an activation of protein kinase A in response to elevated cyclic AMP levels.It is possible that protein kinase A plays a permissive role in allowing nicotinic cholinoceptors to activate TOH by another signalling pathway.

Substance P modulates the time course of nicotinic but not muscarinic catecholamine secretion from perfused adrenal glands of rat.

1. Substance P (SP) and acetylcholine (ACh) are contained within the splanchnic nerve terminals in the adrenal gland and can be released in response to stress. In the rat, the release of aCh brings about secretion of catecholamines (CA) by acting on nicotinic and muscarinic receptors on the adrenal chromaffin cells. 2. In the present study, we have used a rat isolated adrenal gland preparation to investigate the effects of SP, perfused at different concentrations, on CA secretion evoked by 10(-5) M nicotine and 10(-4) M muscarine. 3. In the first 10 min stimulation period (S1), in the absence of SP, nicotine (10(-5) M) evoked substantial and equal secretion of noradrenaline (NA) and adrenaline (Ad). In a second 10 min stimulation period (S2), carried out 18 min after S1, the nicotinic response was desensitized. In contrast, the muscarinic response, which preferentially evoked Ad secretion in S1 (Ad/NA: 8.7/1), was well maintained in S2. 4. SP present in S1 had no effect on desensitization of the subsequent nicotinic response in S2. 5. At low concentrations (10(-7)-10(-10) M), SP changed the time course of nicotine-induced CA secretion during S1 by enhancing CA secretion in the first 4 min and inhibiting CA secretion thereafter. The maximal effect occurred at 10(-9) M SP. 6. At a higher concentration (10(-5) M), SP inhibited total nicotinic CA secretion throughout S1 and produced a biphasic secretion of CA (depressed in the presence of SP and enhanced after wash out of SP). Pre-exposure of adrenal glands to SP (10-' to 10- M) for 10min produced marked inhibition of the nicotine-induced CA secretion. 7. In contrast to the effect of SP on the nicotinic response, SP from 10- to 10-SM had no effect on muscarinic CA secretion. 8. This difference in sensitivity of the nicotinic and muscarinic responses to SP points to a diversity of mechanisms available for control of adrenal catecholamine secretion. In addition to the ability of SP to increase or decrease the total amount of adrenal CA secretion, dependent on the concentration of SP, the present study shows that SP can change the time-course of nicotinic CA secretion. These results with the rat adrenal gland perfused in vitro suggests both a quantitative and temporal role for SP as a novel modulator of adrenal CA secretion.

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 microM) 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 microM. 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 microM histamine was reduced by 45% by 1 microM mepyramine and by almost 30% by 1 microM cimetidine, and was abolished by the combination of both antagonists. Cimetidine at 100 microM did not inhibit the response to histamine more than 1 microM cimetidine. The H3-receptor antagonist, thioperamide (1 microM), had no effect on the response to histamine. 4. The H1-receptor agonist, 2-thiazolyethylamine (5-100 microM) and the H2-receptor agonist, dimaprit (5-100 microM), each induced a cyclic AMP response, and gave more-than-additive responses when combined. The H3 agonist (R) alpha-methylhistamine (100 microM) had no effect either on its own or in combination with either the H1 or the H2 agonist. The response to 100 microM 2-thiazolylethylamine was unaffected by cimetidine (100 microM). 5. The cyclic AMP responses to 5 microM histamine, 100 microM thiazolylethylamine and 100 microM 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 microM forskolin, while the responses to histamine and thiazolylethylamine were weakly enhanced.6. The cyclic AMP response to 5 microM histamine was partially reduced in the absence of extracellular Ca2 and the residual response was fully antagonized by 1 microM cimetidine and was unaffected by 1 microM mepyramine.In the absence of Ca2 , the cyclic AMP response to 100 microM thiazolylethylamine was abolished, while that to 100 microM dimaprit was unaffected.7. Reincubation of 5 microM 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 microm 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 H, 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.

Effect of Gd3+ on bradykinin-induced catecholamine secretion from bovine adrenal chromaffin cells.

1. The effects of Gd3+ on bradykinin- (BK-) induced catecholamine secretion, 45Ca2+ efflux and cytosolic [Ca2+] were studied using bovine adrenal chromaffin cells. 2. BK increased secretion in a Ca2+-dependent manner. From 1 - 100 microM, Gd3+ progressively inhibited secretion induced by 30 nM BK to near-basal levels, however from 0.3 - 3 mM Gd3+ dramatically enhanced BK-induced secretion to above control levels. Gd3+ also increased basal catecholamine secretion by 2 - 3 fold at 1 mM. These effects were mimicked by Eu3+ and La3+. 3. Gd3+ enhanced secretion induced by other agonists that mobilize intracellular Ca2+ stores, but simply blocked the response to K+. 4. Gd3+ still enhanced basal and BK-induced secretion in Ca2+-free solution or in the presence of 30 microM SKF96365, however both effects of Gd3+ were abolished after depleting intracellular Ca2+ stores. 5. Gd3+ (1 mM) reduced the rate of basal 45Ca2+ efflux by 57%. In Ca2+-free buffer, BK transiently increased cytosolic [Ca2+] measured with Fura-2. The [Ca2+] response to BK was substantially prolonged in the presence of Gd3+ (1 mM). 6. The results suggest that Gd3+ greatly enhances the efficacy of Ca2+ released from intracellular stores in evoking catecholamine secretion, by inhibiting Ca2+ extrusion from the cytosol. This suggests that intracellular Ca2+ stores are fully competent to support secretion in chromaffin cells to levels comparable to those evoked by extracellular Ca2+ entry. Drugs that modify Ca2+ extrusion from the cell, such as lanthanide ions, will be useful in investigating the mechanisms by which intracellular Ca2+-store mobilization couples to Ca2+-dependent exocytosis.

The effects of neosurugatoxin on evoked catecholamine secretion from bovine adrenal chromaffin cells.

1. The effect of neosurugatoxin (NSTX), a toxin from the Japanese ivory mollusc (Babylonia japonica), on the nicotinic response of bovine adrenal chromaffin cells was examined. 2. NSTX inhibited acetylcholine- and nicotine-induced catecholamine secretion from the cultured cells with an IC50 against 5 microM nicotine of 30 nM. 3. This inhibitory effect was reversible and independent of the presence of agonist. 4. NSTX had no effect on the catecholamine release from cultured cells evoked by 50 mM K+, or 1 microM histamine. 5. NSTX had no effect on the stimulation of phosphatidylinositol metabolism evoked by 100 microM muscarine. 6. These results suggest NSTX may be useful as a nicotinic receptor probe in tissues such as the adrenal and sympathetic ganglia where alpha-bungarotoxin is ineffective.

Regulation of cyclic AMP metabolism in bovine adrenal medullary cells.

The capacity of cultured bovine adrenal medullary cells to metabolize and export cyclic AMP has been studied. Basal cellular cyclic AMP levels were increased 50% by 100 microM 3-isobutyl-1-methylxanthine (IBMX) and rolipram, a class IV (cyclic AMP-specific) phosphodiesterase (PDE) inhibitor. They were not affected by inhibition of class I (Ca2+/calmodulin-dependent), class III (cyclic GMP-inhibited) or class V PDE (cyclic GMP-specific) with vinpocetine or 3-isobutyl-8-methoxymethyl-1-methylxanthine (8-methoxymethyl-IBMX), SK&F 94120, or MB 22,948, respectively, all at 100 microM. Furthermore, only IBMX and rolipram enhanced the cyclic AMP response to 0.3 microM forskolin. Rolipram had an EC50 of < or = 1 microM and was equally effective at 100 microM and 1 mM. IBMX enhanced cyclic AMP levels significantly more at 1 mM than at 100 microM. Neither vinpocetine nor 8-methoxymethyl-IBMX (100 microM) enhanced the Ca(2+)-dependent cyclic AMP response to K+ depolarization. Elevation of cyclic GMP levels with sodium nitroprusside (10 or 100 microM), to activate any cyclic GMP-stimulated class II PDE and to inhibit any cyclic GMP-inhibited class III PDE, also had no effect on basal or forskolin-stimulated cyclic AMP levels. In the presence of IBMX (1 mM), forskolin (5 microM) caused a rapid and large increase in cellular cyclic AMP levels which was maximal after about 5 min and declined slightly over 3 hr. Over this period, extracellular cyclic AMP levels rose almost linearly reaching levels 2-3 times those in the cells. The results indicate bovine adrenal medullary cells have a high capacity for sustained cyclic AMP export. Furthermore, two PDE isozymes appear to degrade cyclic AMP in these cells, a rolipram-sensitive, cyclic AMP-specific, class IV isozyme and a rolipram-insensitive isoform.

Effects of opioid compounds on desensitization of the nicotinic response of isolated bovine adrenal chromaffin cells.

Opioid compounds have been assessed for their ability to modify desensitization of nicotine-induced catecholamine secretion from cultured, bovine, adrenal chromaffin cells. Dynorphin-1-13 and metorphamide produced protection against desensitization of the nicotinic response at concentrations between 1 and 20 microM while etorphine and morphine only produced this effect at 100 microM. The opioid antagonists, naloxone and diprenorphine, at 100 microM mimicked the weak ability of the opioid agonists to protect against nicotinic desensitization. All opioid compounds tested were considerably more potent at inhibiting nicotine-induced catecholamine secretion from the cells than at protecting against desensitization of this response. It is concluded that adrenal opioid peptides probably do not act on adrenal opioid binding sites characterised from ligand binding studies to prevent the nicotinic response from desensitizing. They are unlikely, therefore, to be involved in such a mechanism to maintain catecholamine secretion during stress.

Effects of opioid compounds on basal and muscarinic induced accumulation of inositol phosphates in cultured bovine chromaffin cells.

The mammalian adrenal medulla expresses a variety of both opioid peptides and opioid receptors. The function of this adrenal opioid system is, however, largely unknown. We have examined the ability of a number of opioid compounds to influence basal and muscarinic stimulated accumulation of inositol phosphates in cultured bovine chromaffin cells. Muscarine produced a dose-dependent 1.5-fold increase in total inositol phosphates. This response was sensitive to atropine inhibition. The ten opioid compounds examined were chosen because between them they possess selectivity for all of the identified opioid receptor subtypes. However, none of these opioids in the concentration range 10nM-10 microM had any significant effect on either basal or muscarinic induced total inositol phosphate accumulation. We conclude that it is unlikely that opioid peptides released from either the chromaffin cells themselves or the splanchnic nerve can modulate the inositol phosphate second messenger system within the adrenal chromaffin cells.

Effects of alpha- and beta-adrenoceptor agonists and antagonists on ATP and catecholamine release and desensitization of the nicotinic response in bovine adrenal chromaffin cells.

The effects of a number of alpha- and beta-adrenoceptor agonists and antagonists on the modulation of secretion from bovine adrenal chromaffin cells were investigated. Secretion was induced by nicotine, 56 mM K+, histamine or Ba2+ and was detected by the ATP luciferin-luciferase bioluminescence technique or by the measurement of endogenous catecholamines (CA) by HPLC coupled with electrochemical detection. ATP release from freshly isolated cells by 5 microM nicotine was only weakly inhibited by adrenaline and noradrenaline and even then required high concentrations (greater than 500 microM), while dopamine (1 microM-1 mM) and isoproterenol (100 microM) had no effect. Clonidine (100 microM), oxymetazoline (100 microM), yohimbine (100 microM), and propranolol (5 microM) all produced inhibition of nicotine-induced ATP release with the order of potency:propranolol greater than oxymetazoline greater than clonidine = yohimbine. The inhibitory effect by propranolol could not be reversed by high concentrations of adrenaline or isoproterenol. In chromaffin cell monolayer cultures, all alpha 2-adrenoceptor agents tested (clonidine, oxymetazoline and yohimbine), produced a dose-dependent, Na+-sensitive, non-competitive inhibition of nicotine-induced catecholamine release with little effect on the catecholamine release induced by K+ (56 mM), histamine (10 microM) or Ba2+ (2.2 mM). (+/-)Propranolol caused a similar pattern of inhibition, however, this inhibition was also observed by (+)propranolol, an isomer with little beta-adrenoceptor antagonist activity. The effects of clonidine and propranolol on desensitization of nicotine-induced CA secretion were also investigated. The degree of desensitization of the nicotinic response was dependent on the concentration of nicotine to which the cells were pre-exposed. Desensitization was detected as the decrease in response to a near EC50 concentration of nicotine (5 microM) following pre-incubation of cells to nicotine in the range of 0.3-300 microM. The desensitization had a threshold of 1 microM nicotine and was maximal at 3 microM nicotine in the pre-incubation. Both clonidine (50 microM) and (+/-)propranolol (5 microM) inhibited CA secretion induced by nicotine (0.3 microM-300 microM) during the pre-incubation period. However, regardless of this inhibition of secretion, neither clonidine nor propranolol had an effect on either the onset, or the rate of nicotine-evoked desensitization subsequently observed. These data suggest that inhibition of the nicotinic response and desensitization of the nicotinic response are regulated independently.(ABSTRACT TRUNCATED AT 400 WORDS)