Dog Adrenals Research Articles

Effects of protein kinase C activator and inhibitor on adrenal catecholamine release in response to splanchnic nerve stimulation in anesthetized dogs.

Effects of protein kinase C (PKC) activator and inhibitors on adrenal catecholamine release were examined in anesthetized dogs. Output of epinephrine (EPI) and norepinephrine (NE) was determined from adrenal venous blood by high-performance liquid chromatography (HPLC) with electrochemical detection. All drugs were infused intraarterially (i.a.) into the adrenal gland through the phrenic abdominal artery. Infusion of the PKC activator phorbol-12,13-dibutyrate (PDB 0.1 micrograms/min) increased EPI and NE output during basal state and enhanced increases in catecholamine output induced by splanchnic nerve stimulation (SNS 1 and 3 Hz). These effects of PDB were abolished by the PKC inhibitor staurosporine (SSP 0.3 microgram/min), when both drugs were infused simultaneously. Infusion of SSP (0.1, 0.3, and 1 micrograms/min) caused a dose-dependent inhibition of the SNS-induced increases in EPI and NE output. SNS-induced increases in catecholamine output were also inhibited by another PKC inhibitor polymyxin B (PMB 0.1, 0.3, and 1 micrograms/min) and by the phospholipase C (PLC) inhibitor neomycin (NM 0.3, 1, and 3 mg/min). SSP, PMB, and NM did not affect basal output of EPI and NE. These results suggest that activation of PKC promotes release of adrenal catecholamines and provide indirect evidence that activation of PKC and PLC may be involved in SNS-induced release of catecholamines from dog adrenal gland.

In vivo evidence for adrenal catecholamine release mediated by nonnicotinic mechanism: local medullary effect of VIP.

The aim of the present study was 1) to develop a model in anesthetized dogs in which local infusion of a given substance could be made to the adrenal gland without any systemic effects and 2) to show in this model the potential existence of a nonnicotinic mechanism involved in adrenal catecholamine secretion. Plasma catecholamine concentrations were determined by an high-performance liquid chromatography method. The local infusion into the left adrenolumbar artery (0.5 ml/min for 1 min) of either dimethylphenylpiperazinium (0.3-3.0 micrograms/ml) or vasoactive intestinal peptide (0.1-10.0 micrograms/ml) resulted in dose-dependent increases in both epinephrine and norepinephrine secretions. Neither aortic pressure nor plasma catecholamine levels altered during the drug infusion. The net increases in adrenal catecholamine secretion obtained with dimethylphenylpiperazinium were abolished by the nicotinic blockade with pentolinium (2 mg/ml, 0.5 ml/min for 2 min). However, the net catecholamine responses to vasoactive intestinal peptide remained unaffected in the presence of the same dose of pentolinium. The results suggest that there exists a nonnicotinic mechanism that may be implicated in the local regulation of medullary catecholamine secretion in the dog adrenal gland. This model may be a useful tool for studying local physiological role(s) of various neurotransmitters and neuromodulators in the adrenal secretory function in vivo.

Hyperadrenocorticism caused by bilateral adrenocortical neoplasia in dogs: Four cases (1983-1988)

Summary: Hyperadrenocorticism caused by bilateral adrenocortical neoplasia was diagnosed in 4 dogs. Three dogs had bilateral adrenocortical adenomas, and 1 dog had bilateral adrenocortical carcinomas. The history, physical findings, clinicopathologic abnormalities, and results of acth stimulation and low-dose dexamethasone suppression tests were compatible with diagnosis of hyperadrenocorticism. Adrenocortical neoplasia was differentiated from pituitary-dependent hyperadrenocorticism on the basis of a combination of test results, including lack of suppression of plasma cortisol after being given a high dose dexamethasone (n = 4), undetectable (< 20 pg/ml) plasma endogenous acth concentration (n = 4), identification of a single mineralized adrenal mass by abdominal radiography (n = 2) and abdominal ultrasonography (n = 1), and identification of bilateral nonmineralized adrenal masses by ultrasonography (n = 1). A left adrenal mass was excised from 1 dog. Clinical signs persisted and administration of mitotane was initiated. One dog was treated only with mitotane. Treatment with ketoconazole was attempted in 2 dogs. All dogs died or were euthanatized because of persistent hyperadrenocorticism. Necropsy and histologic evaluation of the pituitary and adrenal tissue confirmed bilateral adrenocortical neoplasia in all dogs. Bilateral adrenocortical neoplasia should be considered as an uncommon, but possible, cause of bilaterally large adrenal glands in dogs with hyperadrenocorticism.

Effects of nifedipine and Bay-K-8644 on the release of catecholamines from the dog adrenal gland in response to splanchnic nerve stimulation.

1. The effects of nifedipine and Bay-K-8644 on the release of adrenal catecholamines were examined in anaesthetized dogs. 2. Splanchnic nerve stimulation (SNS) at 1 and 3 Hz produced frequency-dependent increases in adrenaline (ADR) and noradrenaline (NA) output determined from adrenal venous blood. 3. Neither nifedipine (10 and 30 micrograms/kg, i.v.) nor Bay-K-8644 (10 and 30 micrograms/kg, i.v.) modified the SNS-induced increases in catecholamine output. Basal catecholamine output tended to be increased and decreased by nifedipine and Bay-K-8644, respectively. 4. Nifedipine produced significant decreases in arterial pressure and renal blood flow rate. Bay-K-8644 produced a significant increase in arterial pressure associated with a decrease in renal blood flow rate. 5. These results suggest that dihydropyridine-sensitive calcium channels do not play a major role in adrenal catecholamine release evoked by SNS.

Fatty acids may regulate aldosterone secretion and mediate some of insulin's effects on blood pressure

Experiments in vitro and observation made in humans suggest that some unesterified fatty acids (FA) participate, as inhibitors, in the regulation of aldosterone secretion. Removal of FA from adrenal glomerulosa cells with albumin increases the responses to angiotensin II (AII) and dibutyry1 cyclic AMP. Micromolar concentrations of some FA including arachidonic, oleic, linoleic, eicosapentaenoic, and docosahexaenoic inhibit aldosterone secretion by adrenal glomerulosa cells. Inhibition is specific — some acids like stearic are inactive, and the adrenal fasciculata is relatively resistant to inhibition. Oleic acid rapidly and reversibly inhibits aldosterone secretion by perfused dog adrenals. Observations in vivo suggest a reciprocal relationship between plasma levels of FA and aldosterone: insulin infusion into dogs lowers plasma FA and increases adrenal responsiveness to All; salt infusions into humans increase plasma FA as aldosterone falls; plasma FA are low in low-renin essential hypertension where adrenal responsiveness to All is high; plasma FA are inversely correlated with ratios of aldosterone to renin in black hypertensives; and plasma FA are high in some seriously ill patients whose aldosterone levels are inexplicably low. All receptors and the final step of aldosterone biosynthesis, oxidation at the 18 position, are the adrenal sites most sensitive to FA. Insulin's antinatriuresis may be mediated in part by its ability to lower plasma FA and thereby enhance adrenal response to secretagogues.

Effects of pirenzepine and hexamethonium on adrenal catecholamine release in responses to endogenous and exogenous acetylcholine in anesthetized dogs.

We investigated the way in which hexamethonium and pirenzepine, a selective muscarinic (M) 1 receptor antagonist, modify the release of catecholamines from dog adrenal gland in response to endogenous and exogenous acetylcholine (ACh) in vivo. Output of epinephrine (EPI), and norepinephrine (NE) was determined from adrenal venous blood by high-performance liquid chromatography (HPLC) with electrochemical detection. Intraarterial (i.a.) injections of ACh (1.5 and 3 micrograms) or muscarine (3 and 6 micrograms) into the phrenicoabdominal artery and splanchnic nerve stimulation (SNS, 3 Hz) produced marked increases in EPI and NE output. Hexamethonium (1 mg/kg intravenously, i.v.) inhibited the increases in EPI and NE output in response to exogenous ACh and SNS. Pirenzepine (10 micrograms/kg i.v.) inhibited the ACh-induced increases in EPI and NE output but did not modify the SNS-induced increases. The muscarine-induced increases in EPI and NE output were also inhibited by pirenzepine. These results indicate that the exogenous ACh-induced release of adrenal catecholamines is mediated through both nicotinic and M1 receptors, in contrast to the predominant mediation of nicotinic receptors in response to endogenous ACh.

Facilitatory Role of the Renin-Angiotensin System in Controlling Adrenal Catecholamine Release in Hemorrhaged Dogs

Effects of the renin-angiotension system (RAS) on adrenal catecholamine release in response to hemorrhagic hypotension and splanchnic nerve stimulation (SNS) were studied in pentobarbital-anesthetized dogs. In hemorrhage experiments, mean blood pressure (MBP) was maintained at 50 mm Hg for 60 min by bleeding the arterial blood into a pressurized bottle. In the renal intact group (control), epinephrine (EPI) and norepinephrine (NE) output from the adrenal gland increased markedly during hemorrhagic hypotension: from 45 +/- 13 and 4.7 +/- 0.9 to 1,167 +/- 202 and 169 +/- 30 ng/min at 60 min after onset of hemorrhage, respectively. The increases in catecholamine output during hemorrhagic hypotension in the renal-intact group pretreated with captopril (1 mg/kg intravenously, i.v.) and in the renal-ligated group were significantly smaller than those in the control group. The increases in catecholamine output in the renal-ligated group infused with angiotensin II (AngII 10 ng/kg/min i.v.) were comparable to those in the control group. In SNS experiments, AngII infusion (10 ng/kg/min i.v.) enhanced increases in catecholamine output induced by 3 Hz SNS significantly. Captopril (1 mg/kg i.v.) did not affect the SNS-induced increases in catecholamine output. These results suggest that the renal RAS facilitates reflex release of adrenal catecholamines during hemorrhagic hypotension, at least in part, by acting directly on the release process of catecholamines from dog adrenal gland.

Nicotinic- and muscarinic-evoked release of canine adrenal catecholamines and peptides

The tissue content and overflow of norepinephrine (NE), epinephrine (Epi), dopamine (DA), Met-enkephalin (Met-Enk), and neuropeptide Y (NPY) from isolated, retrogradely perfused dog adrenal glands were studied. Under resting conditions, approximately 25% of the overflow of autocoids from the glands was Ca2+ dependent; the cholinergic antagonists hexamethonium and atropine had no effects on basal efflux. Stimulation with the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP; 3 or 50 microM) or with the muscarinic agonist pilocarpine (50 microM or 1 mM) evoked releases of autocoids. These releases were blocked or dramatically reduced by appropriate antagonists or by the removal of Ca2+ from the perfusate. Expressed as percentages of tissue stores, the rank order of overflow of autocoids was E approximately DA much greater than NE during resting conditions, DA much greater than E approximately NE during stimulation with 50 microM DMPP, and DA greater than Epi greater than NE during stimulation with 1 mM pilocarpine. These data are consistent with different mechanisms of release for the catecholamines, perhaps from different cell populations. The data support corelease of peptides and catecholamines, although clear pairing of autocoids could not be confirmed.

Changes in lipid peroxidation induced by chloditane in adrenals of dogs and guinea pigs

Changes in lipid peroxidation induced by chloditane in adrenals of dogs and guinea pigs

Structural aspects of cerebral cortical synaptic function in the early postresuscitation period

The effect of adrenocorticolytic drug chloditan (1-(o-chlorophenyl)-1-(p-chlorophenyl)-2,2-dichloroethane;) on the content and the rate of lipid peroxidation in dog and guinea pig adrenals was studied. In adrenals of dogs and guinea the amount of malondialdehyde (MDA) was increased. In vitro spontaneous lipid peroxidation was delayed in adrenal homogenate of DDD-treated dogs. Spontaneous lipid peroxidation in adrenal homogenate was not decreased by feeding DDD in the guinea pig. DDD consumption did not affect on lipid peroxidation promoted by ascorbic acid and ferrous iron. The accumulation of MDA in dog and guinea pig adrenal homogenates was inhibited by NADPH.

Cholesterol increase in mitochondria: a new method of cholesterol incorporation.

In this study, we report a new method for cholesterol incorporation in mitochondria. This method is based on the coupling of cholesterol to bovine serum albumin (BSA) to form a structure with a spherical shape (designated C-BSA-C). The results show that C-BSA-C induces a specific cholesterol incorporation in mitochondria without interference from BSA. The cholesterol increase is complete in 1 minute, and is dose-related and pH-dependent. Temperature also has an effect. In mitochondria from dog adrenal glands, pregnenolone biosynthesis induced by C-BSA-C is increased, indicating that only cholesterol is transferred to the membrane.

A study of non-aromatizing androgen 19-hydroxylase in sheep adrenal

We reported on the unusually high isotope effect of non-aromatizing androgen 19-hydroxylase in sheep and dog adrenals and the validity of the [3H] water method using [19-3H3] androgen. We have extended the study to examine whether this 19-hydroxylation is catalyzed by a cytochrome P-450 dependent enzyme. Sheep adrenal homogenate (1.65 mg prot.) was incubated in the presence of NADPH (5.6mM) with [19-3H3, 4-14C]-androstenedione (A) (3.2 microM, 8.24 x 10(4) dpm 3H/micrograms, 3H/14C = 17.2) in a total of 1.2 ml PO4 buffer under air at pH 7.4 for 2, 5 and 10 min. [19-3H2, 4-14C]-19-hydroxy-A (19-OHA) with added carrier was purified through extraction, TLC, acetylation to form 19-AcOA, and further TLC to give 19-hydroxylase activity as assessed by the product isolation method. Simultaneously, the [3H] water was measured by distillation, and with correction by the apparent kinetic isotope effect (KH/KT = 11.8), used for assessment of 19-hydroxylase activity. The effects on the hydroxylation by cofactor (NADPH, NADH), incubation atmosphere (N2, CO/O2), cytochrome P-450 inhibitors (metyrapone, clotrimazole) and heating were measured by both methods. Compared to the complete system (89.6pmol/min/mg as 100%), carbon monoxide suppressed 15.8, 59.3 and 86.4% of the 19-hydroxylation when a CO/O2 ratio of 0.1, 1 and 9 was used, respectively. Replacement to nitrogen atmosphere decreased the activity by 93.8%. Replacement of NADPH with NADH (7.5mM) caused more than a 92.1% decrease in activity. Metyrapone at 50 and 200 microM and and clotrimazole at 2.5 and 10 microM suppressed the activity by 82.8, 90.4, 85.4 and 94.9%, respectively. A larger scale sheep adrenal incubation of A (250 microM) under 18O2 atmosphere and isolation of 19-AcOA were carried out in a similar manner. The gas chromatography-mass spectrometry analysis of the purified product showed 48.5% of the product to be 18O-labeled as [M+ + 2], m/e 346. Thus, the non-aromatizing androgen 19-hydroxylase requires NADPH and molecular oxygen. It is strongly inhibited by carbon monoxide and cytochrome P-450 inhibitors. These results indicate that the enzyme system responsible for non-aromatizing androgen 19-hydroxylase in adrenal is a cytochrome P-450 dependent monooxygenase.

A modulating role of prostaglandins in catecholamine release by perfused dog adrenal glands

PGE 2, PGE 1, PGF 2α and PGD 2 dose dependently potentiated the ACh-evoked catecholamine (CA) release from perfused dog adrenal glands. These prostaglandins by themselves slightly stimulated CA release. PGE 2 potentiated the CA release induced by stimulation of nicotinic or muscarinic cholinergic receptors and also by stimulation with excess K + or caffeine. However, PGE 2 did not affect the release induced by caffeine. However, PGE 2 did not affect the release induced by caffeine in the absence of extracellular Ca 2+. ACh stimulated the 45Ca efflux from adrenals prelabelled with 45Ca. PGE 2 enhanced the ACh-stimulated 45Ca efflux. The CA release in response to ACh or nicotine was slightly but significantly reduced by pretreatment of the adrenals with indomethacin. SC19220, a specific prostaglandin antagonist significantly reduced the ACh-evoked CA release and blocked the enhancing action of PGE 2 on the stimulation-evoked CA release. The results showed that prostaglandins stimulated the CA release evoked in perfused dog adrenals and that the effect on the Ca 2+ flux across the plasma membrane could be involved in the prostaglandin mechanism of action. The possibility is suggested that prostaglandins formed endogenously in response to physiological stimulation of chromaffin cells could play a facilitating, modulatory role in CA release.

Impaired adrenal vascular response to hemorrhagic hypotension by an adreno-femoral venous shunt

We studied the influence of an adreno-femoral venous shunt on the regional blood flow in the dog adrenal gland during hemorrhagic hypotension. An adreno-femoral venous shunt was placed transperitoneally in the left adrenal gland while the animal was under pentobarbital anesthesia. The right adrenal gland was left intact. Hemorrhagic hypotension was induced at 50 mm Hg mean arterial blood pressure for 1 hr in six dogs and the other six dogs served as normotensive controls. Nonradioactive microspheres (2 × 10 6, 15 ± 2 μm) were injected into the ventricle. The adrenal cortical and medullary blood flows were estimated by microscopic counting of the microspheres in the serially sectioned adrenal gland. The total adrenal blood flow (sum of the cortical and medullary blood flows) of two methods was compared; the microsphere method vs timed venous sampling through the shunt. In the intact right adrenal gland, the cortical blood flow was significantly decreased, but the medullary blood flow remained unchanged during hemorrhagic hypotension. Subsequently the total adrenal blood flow was not significantly decreased. In the cannulated left adrenal gland, both the cortical and medullary blood flows were significantly decreased and adrenal vascular autoregulation was inhibited. The total adrenal blood flow calculated by the microsphere method showed a significant correlation with that by timed venous sampling through the shunt. These results indicate that the adrenal medullary vasculature autoregulates during hemorrhagic hypotension and that an adreno-femoral venous shunt abolishes this vascular response.

Comparison of corticotropin-releasing factor and acetylcholine on catecholamine secretion in dogs.

To test whether corticotropin-releasing factor (CRF) is a secretagogue for adrenal secretion of catecholamines, a preparation was developed that permits measurement of adrenal venous output in response to in vivo arterial injection into the dog adrenal gland. Dogs were prepared with catheters in the lumboadrenal vein for monitoring adrenal blood flow and secretion rate of epinephrine and norepinephrine and in the lumboadrenal and inferior phrenic arteries for adrenal injection. Under pentobarbital anesthesia 48 h after surgery, dogs received a series of intra-adrenal injections that included acetylcholine (0.2-200 nmol), CRF (2.0-20.0 nmol), and diluent. There was a log dose-response relationship before epinephrine secretion and norepinephrine secretion to acetylcholine. Adrenal injection of CRF stimulated epinephrine and norepinephrine secretion at the highest dose tested (20 nmol). The response observed was equivalent to the response to 0.2 nmol acetylcholine. A similar dose of CRF given systemically produced hypotension without stimulating catecholamine responses. Adrenal catecholamine responses to acetylcholine were not augmented by addition of CRF. These findings show that arterial injection of CRF into the intact dog adrenal stimulates secretion of epinephrine and norepinephrine. However, the low potency of CRF relative to that of acetylcholine, the lack of a synergistic effect of CRF on catecholamine responses to acetylcholine, and the high dose of CRF required to achieve a response suggest that CRF does not function in the adrenal medulla as a physiologically important secretagogue for catecholamines.

Forskolin enhancement of acetylcholine-evoked cyclic AMP formation and catecholamine release in perfused dog adrenals.

Unstimulated efflux of cyclic AMP from perfused dog adrenal glands was not altered by 0.1 microM of forskolin and was slightly increased by 0.3 and 1.0 microM of forskolin. ACh stimulated efflux of cyclic AMP which preceded CA release and the efflux was dose-dependently enhanced by forskolin. Forskolin did not affect the spontaneous CA release but enhanced ACh-evoked catecholamine (CA) release. There was a close correlation between the dose relationship of forskolin enhancement of stimulated-cyclic AMP efflux and that of evoked-CA release. ACh-evoked CA release in the presence of forskolin was further potentiated by R020-1724, a phosphodiesterase inhibitor. CA release evoked by excess K+, or by caffeine in the presence or absence of external Ca2+ was also potentiated by forskolin. These results suggests that cyclic AMP generation may increase in response to stimulation of adrenal chromaffin cells and that the resulting increase of the nucleotide may function as a facilitating modulator of CA release.

Stimulation-Evoked Cyclic Nucleotides Efflux from Isolated Perfused Dog Adrenals and Possible Involvement of Calcium

Nicotine and muscarine caused the transient increase in cAMP and cGMP efflux from dog adrenal glands followed by a small but lasting increase in the nucleotide efflux. The initial increase preceded catecholamine (CA) release and the latter slowly developed. Nicotine and muscarine caused the maximal increase of cAMP and cGMP levels in adrenal medulla 15 sec after the treatment, and these effects were antagonized by hexamethonium and atropine, respectively. Hexamethon um in combination with atropine, verapamil and an omission of Ca2+ in the medium prevented ACh from producing the increase in cyclic nucleotide efflux and CA release. Reintroduction of Ca2+ (1.3 mM) in fluid after perfusion with Ca2+ and Mg2+-free fluid caused the transient increase in cyclic nucleotide efflux and CA release. Adenylate cyclase activity in adrenal medulla was activated by Ca2+. These results may suggest that cAMP formation was increased by activation of adenylate cyclase as a result of increased influx of Ca2+ when adrenal medulla were stimulated.

Potassium ion is indispensable to the catecholamine releasing response of dog adrenals to gamma-aminobutyric acid.

The effect of potassium ion on the GABA-evoked catecholamine (CA) release from isolated perfused adrenal glands of the dog was investigated. When omitting the external potassium ion, the basal release of CA was increased. During this period GABA no longer caused the increase in CA release and moreover the increased basal release was diminished reversibly by GABA. 3-Amino-1-propane-sulfonic acid, a GABAA agonist, mimicked the action of GABA in K+-free solution, while baclofen, a GABAB agonist, did not cause CA release in normal solution and did not alter the basal release in K+-free solution. The inhibition by GABA of the basal CA release in K+-free solution was blocked by bicuculline. The potency of the CA releasing action of GABA was dependent on the concentration of external K+ between 1-10 mM. Reintroduction of K+ to glands which had been perfused with K+-free solution immediately reduced the basal release of CA whereas it recovered the CA releasing action of GABA. These results suggest that GABA-evoked CA release is dependent on potassium ion. The possible mechanisms by which GABA evoked CA release are discussed.

O2E1312) - Possible involvement of potassium ion in catecholamine release from isolated perfused dog adrenals evoked by GABA.

O2E1312) - Possible involvement of potassium ion in catecholamine release from isolated perfused dog adrenals evoked by GABA.

The nature of the stimulatory action of gamma-aminobutyric acid in the isolated perfused dog adrenals.

The effect of gamma-aminobutyric acid (GABA) on catecholamine (CA) release from adrenal medulla was investigated. GABA and GABA agonists, 3-amino-1-propane-sulfonic acid and imidazole-4-acetic acid caused CA release from isolated perfused dog adrenals in a dose-dependent manner, and no tachyphylaxis to GABA was observed. CA release elicited by GABA was antagonized by bicuculline and picrotoxin. This antagonism was specific for GABA- and GABA agonist-induced responses, response to acetylcholine being unaffected. Pretreatment with atropine plus hexamethonium did not affect the response to GABA. GABA-induced CA release was abolished by the removal of Ca2+ from perfusion medium, but not by the removal of Na+ or Cl-. Verapamil, CoCl2 and dibucaine blocked the effect of GABA. A Na+ channel blocker, tetrodotoxin did not reduce GABA-evoked CA release. These results suggest that GABA may interact with its receptor to evoke CA release from adrenal medulla in a fashion of Ca2+-dependence and independence on external Na+ or Cl-.