The arterial supply and venous drainage of 62 left and 5 right ovine adrenal glands is described, and the contribution of individual arteries to successful adrenal gland autotransplantation was evaluated. Arterial flow was measured by direct collection from the draining adrenal vein. Assessment of function of the transplanted adrenal gland was made from survival of the sheep and by the cortisol response to infusion of ACTH and the aldosterone secretory response to infusion of angiotensin II or potassium. For the left adrenal, the principal arterial supply was from the renal artery in 21 (34%), a lumbar artery in 32 (52%), and the anterior mesenteric artery in 3. The total blood flow was 5.0 +/- SEM 0.4 mL/min, the flow from the renal branch 2.3 +/- 0.3 mL/min, and the principal lumbar branch 2.6 +/- 0.3 mL/min. Venous drainage from the left adrenal was via a major adrenal vein to the left renal vein, but additional tributaries to the renal vein were present in 26%. The arterial supply to the adrenal is regional and omission of a branch at transplantation could result in infarction of portion of the gland. By defining arterial supply and measuring blood flow, selection of the appropriate artery or multiple arteries can achieve an adrenal gland autotransplant survival of 90%.

A specific and sensitive radioimmunoassay has been developed for the measurement of the secreted carbonic anhydrase isoenzyme (CA VI) in sheep saliva and tissues. The assay can detect as little as 75 pg of CA VI, and the antibody used does not cross-react with CA II or CA III. The intra-assay variation, measured using a saliva sample, was 3.0%, whereas the inter-assay variation was 10.5%. The concentration of CA VI in parotid saliva from normal, resting sheep was 5.6 +/- 3.0 micrograms.ml-1 (n = 42) or 79.4 +/- 35.7 micrograms.mg of total protein-1. With feeding, the CA VI concentrations increased an average of 6-fold. The secretion rate of CA VI from the vascularly isolated neurotomized parotid gland of the anaesthetized sheep was 0.62 +/- 0.40 micrograms.min-1, compared with a rate of 11.7 +/- 7.8 micrograms.min-1 from the parotid gland of normal conscious sheep. Stimulation of the parotid-gland preparation by the muscarinic agent bethanechol increased the secretion rate to 438 +/- 172 microgram.min-1 (n = 8), and electrical stimulation of the secretomotor Moussu nerve increased CA VI secretion rate to 634 +/- 330 micrograms.min-1 (n = 4). Submandibular saliva from anaesthetized sheep contained 6.9 +/- 2.1 micrograms of CA VI.ml-1 (n = 3). The only tissues found to contain measurable amounts of CA VI were the parotid (6.4 micrograms.mg of protein-1) and submandibular (1.8 micrograms.mg of protein-1) salivary glands. The sublingual salivary gland, kidney, lung, adrenal, brain, skeletal muscle, liver, heart, pancreas, small intestine and cerebrospinal fluid did not have a measurable CA VI content.

1. The proposition that stimulation of the secretomotor nerve to the ovine parotid gland might involve co-release of vasoactive intestinal peptide (VIP) was tested by studying responses to infusion of VIP directly into the gland's arterial blood supply and by assay of VIP in parotid venous blood.2. In unstimulated glands, an arterial blood concentration of 1.5-2.5 x 10(-9) mol/L VIP did not evoke fluid secretion but it increased K+ and phosphate secretion and glandular blood flow. The same blood concentration of VIP potentiated the stimulation of salivary flow rate caused by intra-arterial infusion of bethanechol but nerve stimulation was not potentiated. VIP increased glandular blood flow in both conditions of stimulation.3. Atropine blocked neurally stimulated salivary secretion but an increase in glandular blood flow was still detectable. There was therefore no evidence for a non-cholinergic neural mechanism for salivary secretion.4. Furthermore, VIP concentrations in glandular venous blood were not increased by nerve stimulation.5. The results indicate that exogenous VIP can affect the flow and composition of ovine parotid secretion but was not involved in the response to secretomotor nerve stimulation.

Complementary DNA clones coding for the human secreted carbonic anhydrase isozyme (CA VI) have been isolated and their nucleotide sequences determined. These clones identify a 1.45-kb mRNA that is present in high levels in parotid submandibular salivary glands but absent in other tissues such as the sublingual gland, kidney, liver, and prostate gland. Hybridization histochemistry of human salivary glands shows mRNA for CA VI located in the acinar cells of these glands. The cDNA clones encode a protein of 308 amino acids that includes a 17 amino acid leader sequence typical of secreted proteins. The mature protein has 291 amino acids compared to 259 or 260 for the cytoplasmic isozymes, with most of the extra amino acids present as a carboxyl terminal extension. In comparison, sheep CA VI has a 45 amino acid extension [Fernley, R. T., Wright, R. D., & Coghlan, J. P. (1988b) Biochemistry 27, 2815]. Overall the human CA VI protein has a sequence identity of 35% with human CA II, while residues involved in the active site of the enzymes have been conserved. The human sheep secreted carbonic anhydrases have a sequence identity of 72%. This includes the two cysteine residues that are known to be involved in an intramolecular disulfide bond in the sheep CA VI. The enzyme is known to be glycosylated and three potential N-glycosylation sites (Asn-X-Thr/Ser) have been identified. Two of these are known to be glycosylated in sheep CA VI. Southern analysis of human DNA indicates that there is only one gene coding for CA VI.

The vascularly isolated parotid glands of sheep were submaximally stimulated for 4 min by nerve stimulation, or by infusion of acetylcholine (ACh) or bethanechol directly into the artery supplying the gland. The three modes of stimulation caused almost equal increases in the rate of salivary flow, initial losses of K+ and phosphate from the gland and total glandular deficits of K+ and phosphate. Concurrent arterial infusion of the K+ channel blocker tetraethylammonium (TEA), at 1.5-3.3 mM in blood, almost abolished these responses to bethanechol but did not alter the responses to nerve stimulation or ACh. The responses to bethanechol were restored by increasing the concentration fourfold. Concurrent arterial infusion of 4-aminopyridine (4-AP), at 0.1-2.3 mM in blood, partially inhibited the increase in salivary flow due to bethanechol but not the response to nerve stimulation or ACh. The specificity and competitive nature of the action of TEA and 4-AP on responses to direct muscarinic stimulation by bethanechol are consistent with blockade of K+ channels in secretory end-piece cells. The failure of TEA and 4-AP to inhibit responses to nerve stimulation and ACh may be due to the blocking agents potentiating the release of neurotransmitter ACh. It is also possible that nerve stimulation and ACh may cause the release of a co-transmitter which bethanechol does not.

The gene encoding the human secreted carbonic anhydrase isozyme CAVI(CA6) maps to chromosome 1 by Southern analysis of a somatic cell hybrid panel and to 1p36.22----p36.33 by in situ hybridization. CA6 is therefore not linked to the cytoplasmic carbonic anhydrase genes on chromosome 8 or to CA7 on chromosome 16.

The effects of Ca2+-active agents on ovine salivary flow rate and composition were measured in parotid glands under several conditions: no stimulation, submaximal stimulation of the secretomotor nerve, or stimulation by the cholinergic agents carbachol or bethanechol. Agents were infused into the arterial blood supply of parotid glands and those investigated were: calcium chloride, the calcium ionophores Bay K8644 and A23187, the calcium chelators EGTA and EDTA, the voltage-dependent calcium channel blocker verapamil and congeners, the calmodulin inhibitor trifluoperazine (TFP) and congeners. None of the agents affected the flow rate of saliva from unstimulated or pharmacologically stimulated glands. Increased plasma [Ca2+] and the ionophores did not affect salivary flow in nerve-stimulated glands. In nerve-stimulated glands, EGTA and TFP reduced salivary flow rate and verapamil increased it. The effect of EGTA was reversed by restoring plasma [Ca2+] to normal (1.0-1.2 mmol/l) or above, but the responses to TFP and verapamil were not reversed by increasing plasma [Ca2+]. In all three conditions of stimulation, infusions of EGTA, verapamil or TFP increased salivary [HPO4(2-)] and reduced [HCO3-] and pH. The ionophores had the opposite effects but increased plasma [Ca2+] had no effect. At the same time, EGTA, verapamil or TFP increased salivary [Na+ + K+], Bay K8644 had the opposite effect but increased plasma [Ca2+] had no effect. The osmolality of the saliva was not altered in any of these circumstances. Salivary [Ca2+] was increased by Ca2+ infusion and reduced by EGTA. Glandular blood flow increased with infusion of agents which increased salivary [HPO4(2-)], fell with infusion of ionophores, and was unchanged by increased plasma [Ca2+]. Thus, there appear to be three calcium-related activities in ovine parotid salivary gland in vivo: (1) salivary flow rate by action at the neuroeffector site, (2) salivary composition by alteration of the ratio of HPO4(2-): HCO3-, and (3) rate of blood flow through the gland by altering vascular resistance.

The secreted carbonic anhydrases, CA VI, are high molecular mass, oligomeric enzymes originally found in the sheep parotid gland and saliva. The enzymes have been purified from the saliva or parotid glands of several different species. All the CA VI enzymes studied have an apparent subunit Mr of about 45,000 as previously reported for the sheep enzyme. By Western analysis, CA VI from human, cow and dog cross-reacted with antibody raised against the purified sheep enzyme whereas that of the mouse did not. The N-terminal sequences of the sheep, human, cow and mouse enzymes are reported. The sheep, cow and human N-terminal sequences are similar to one another while the mouse sequence is substantially different. Nevertheless, the amino acids in the aromatic cluster I (Trp-5, Tyr-7, Trp-16 and Tyr/Phe-20) have all been conserved, as is the case with the cytoplasmic carbonic anhydrases. Eighteen tissues from the sheep have been examined for the presence of CA VI by Western analysis but it has been found only in the salivary glands. Northern analysis and hybridization histochemistry show that the mRNA for CA VI in sheep is expressed specifically in the acinar cells of the parotid and submandibular glands.

Approximately half the carbonic anhydrase activity of sheep parotid-gland homogenate is derived from a high-Mr protein [Fernley, Wright & Coghlan (1979) FEBS Lett. 105, 299-302]. This enzyme has now been purified to homogeneity, and its properties were compared with those of the well-characterized sheep carbonic anhydrase II. The protein has an apparent Mr of 540,000 as measured by gel filtration under non-denaturing conditions and an apparent subunit Mr of 45,000 as measured by SDS/polyacrylamide-gel electrophoresis. After deglycosylation with the enzyme N-glycanase the protein migrates with an apparent Mr of 36,000 on SDS/polyacrylamide-gel electrophoresis. The CO2-hydrating activity was 340 units/mg compared with 488 units/mg for sheep carbonic anhydrase II measured under identical conditions. This enzyme does not, however, hydrolyse p-nitrophenyl acetate. The enzyme contains 0.8 g-atom of zinc/mol of protein subunit. The peptide maps of the two carbonic anhydrases differ significantly from one another, indicating they are not related closely structurally. Unlike the carbonic anhydrase II isoenzyme, which has a blocked N-terminus, the high-Mr enzyme has a free glycine residue at its N-terminus.

The primary structure of the secreted carbonic anhydrase from ovine salivary glands has been determined by automated Edman sequence analysis of peptides generated by cyanogen bromide and tryptic cleavage of the protein and Staphylococcus aureus V8 protease, trypsin, and alpha-chymotrypsin subdigests of the large cyanogen bromide peptides. The enzyme is a single polypeptide chain comprising 307 amino acids and contains two apparent sites of carbohydrate attachment at Asn-50 and Asn-239. The protein contains two half-cystine residues at 25 and 207 which appear to form an intramolecular disulfide bond. Salivary carbonic anhydrase shows 33% sequence identity with the ovine cytoplasmic carbonic anhydrase II enzyme, with residues involved in the active site highly conserved. Compared to the cytoplasmic carbonic anhydrases, the secreted enzyme has a carboxyl-terminal extension of 45 amino acids. This is the first report of the complete amino acid sequence of a secreted carbonic anhydrase (CA VI).

The role of Ca2+ in stimulation of aldosterone secretion (ASR) has been evaluated in vivo using conscious sheep with an adrenal cervical autotransplant. The calcium antagonists verapamil, nisoldipine, and lanthanum and the calcium ionophore BAY K 8644 were infused into the adrenal arterial supply before or concomitantly with angiotensin II. Nisoldipine reversed stimulation of ASR (n = 4) from 13.6 +/- 3.2 to 4.8 +/- 1.2 nmol/h (P less than 0.01; control 2.3 +/- 0.6 nmol/h), as did verapamil. Lanthanum had an intermediate effect. In contrast, pretreatment with nisoldipine (n = 5) did not affect the response to angiotensin II, with ASR being 3.8 +/- 0.9 nmol/h after nisoldipine alone and 12.8 +/- 1.3 nmol/h after nisoldipine plus angiotensin II. In response to graded infusion of angiotensin II, nisoldipine blunted (P less than 0.01) to a small degree the response at all doses of the peptide. Close adrenal arterial infusion of the ionophore BAY K 8644 similarly reversed stimulation of ASR by angiotensin II. It also blocked the initiation of response to the peptide. These data are consistent with the involvement of two pools of calcium in the zona glomerulosa response to angiotensin II, an intracellular pool that is primarily responsible for the initiation of response and a transmembrane extracellular pool that is primarily involved in the "sustained" response to angiotensin II.

SUMMARY 1. To investigate a role for peptides derived from the precursor molecule pro‐ opiomelanocortin (POMC) on the control of aldosterone secretion (ASR), α‐, β‐, γ 1 , and γ 2 ‐melanocyte stimulating hormone (MSH), corticotropin‐like intermediate lobe peptide (CLIP) or β‐endorphin were infused into the adrenal arterial supply of sheep with an adrenal cervical autotransplant. 2. None of the peptides had any significant effect on aldosterone secretion rate in Na replete, unstressed, conscious animals. In contrast, ACTH‐stimulated ASR approximately twofold. 3. POMC‐derived peptides other than ACTH appear to have little or no effect on the short‐term control of aldosterone secretion in vivo , although a role in control and modulation of adrenal function over the longer term cannot be discounted.

Secretion by the parotid gland of Na-replete and -depleted sheep was investigated by examining the effects of modifiers of ionic transfer on salivary composition and flow rate. These agents were infused into the arterial blood supply of the vascularly isolated gland in anesthetized sheep. Ouabain inhibited Na+-K+ exchange in the ducts caused by Na depletion and restored the [Na+], [K+], and osmolality to close to those of Na-replete saliva. Ouabain also inhibited Cl- -HCO3- exchange in the ducts in Na repletion and depletion. Amiloride partially inhibited Na+-K+ exchange in Na depletion without affecting Cl- -HCO3- exchange. Monensin potentiated Na+-K+ exchange in Na repletion and depletion. Amiloride and monensin gained access to the saliva, but furosemide and ethacrynic acid were almost totally excluded, and, up to 10(-3) M in blood, they did not affect salivary composition or flow rate. Methazolamide gained free access to saliva but was without effect. 4-Acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid at 10(-3) M slightly increased salivary [Na+] and [HPO4(2-)]. The results indicate potent effects of ouabain on basolateral Na+-K+ pumps and of amiloride and monensin on transcellular delivery of Na+ to these pumps, but ouabain had no effect on salivary flow rate until O2 consumption approached zero and secretion failed. The findings do not support a proposal that the salivary secretion depends on a Cl- -dependent furosemide-sensitive system energized by Na+-K+-ATPase-dependent Na pumps.

Conscious sheep whose sole adrenal gland had been transplanted to the neck to allow access to the adrenal vasculature were used to study the effect of calcium antagonists on aldosterone secretion. All animals were sodium depleted by uncompensated loss of parotid saliva. The drugs EDTA, verapamil, methoxyverapamil, nisoldipine, lanthanum, propylmethylenedioxyindene, and ryanodine were infused on separate occasions in three or four increasing dose levels. All infusions were made to produce known concentrations directly into the adrenal arterial blood supply. None of these infusions had any significant effect on aldosterone secretion rate, cortisol secretion rate, and little or no effect on plasma [Na], [K], or blood pressure. At high infusion rates some agents (verapamil, methoxyverapamil) caused tachycardia. In contrast, angiotensin II stimulation of aldosterone secretion is inhibited by both verapamil and nisoldipine. The data demonstrate that the sustained elevation of aldosterone secretion caused by sodium depletion is not dependent on a sustained alteration in transmembrane calcium flux. Furthermore, if circulating angiotensin II is the primary stimulus to aldosterone during sodium depletion, its mechanism of action appears to switch to one which is not dependent on calcium alone.

The handling of phosphate by the sheep's parotid gland was investigated by examining the effects on the composition of saliva of phosphate depletion, secretomotor nerve stimulation, enrichment of phosphate in the blood supply, and parotid arterial infusion of parathyroid hormone (PTH) and calcitonin. Experiments were performed in anesthetized sheep after vascular isolation of the parotid gland. In phosphate depletion arterial plasma phosphate fell from 1.50 +/- 0.12 to 0.73 +/- 0.08 mM. In phosphate repletion salivary phosphate ion was concentrated 8- and 16-fold compared with parotid arterial and venous plasma, respectively. In depletion salivary phosphate concentration was unchanged at slow salivary flow rate but was significantly lower at high flow rate. Enrichment of phosphate in the arterial blood increased salivary phosphate concentration in both situations. Synthetic bPTH (1-34), highly purified bPTH (1-84), and bovine parathyroid extract were equipotent in increasing salivary phosphate concentration in phosphate repletion and had no effect in phosphate depletion. They also increased parotid blood flow. The rise in phosphate with PTH was conditional on the rise in blood flow, but the same rise in blood flow alone did not reproduce the effect of PTH on salivary phosphate. PTH increased work done and oxygen consumption by the gland but arterial phosphate enrichment did not. Increases in salivary phosphate were associated with decreases in salivary bicarbonate, and the sum [HCO-3] + [Cl-] + (HPO2-4] remained almost constant. Micropuncture studies have shown that ovine parotid ducts do not normally reabsorb phosphate and therefore PTH probably increases phosphate secretion in the endpieces.

Infusion of bovine parathyroid hormone (bPTH) preparations into the arterial blood supply of the vascularly isolated parotid gland in anaesthetized sheep increases salivary phosphate concentration and gland blood flow rate with rapid onset and offset of action. These responses have been used as a bioassay for PTH and PTH analogues and for assessing the properties of an in-vitro inhibitory analogue [Nle-8, Nle-18, Tyr-34]bPTH-(3-34)amide. [Nle-8, Nle-18, Tyr-34]bPTH-(1-34)amide at 10(-9) to 10(-8) mol/l was four to five times more potent than bPTH(1-34) on both salivary phosphate and blood flow assays. Human PTH(1-34) was not significantly more potent than bPTH(1-34). The [Nle-8, Nle-18, Tyr-34]bPTH-(3-34)amide analogue had very slight agonist activity at 3 X 10(-7) mol/l and at a 100:1 ratio of analogue to PTH it completely inhibited the action of bPTH(1-34) on phosphate secretion and gland blood flow. It caused partial inhibition at 10:1 and had no evident effect at 1:1. These results differ from previous in-vitro results and indicate that the preparation may be valuable for evaluation of agonist and antagonist analogues of PTH. The vascularly isolated parotid gland of the sheep permits repeated random testing of analogues in a control-test-control sequence and the results indicate high sensitivity to PTH in a rapidly reactive in-vivo system with two responding parameters.