Biomedical subjects

A I Smith

Publications and source records attributed to A I Smith.

At least 73 records · Page 4Linked to original sources

PubMed1993

Identification and characterization of an amidating enzyme in ovine heart.

1. Levels of peptidylglycine alpha-amidating mono-oxygenase (PAM) activity were examined in sheep and rat heart. This enzyme is responsible for alpha-amidation of a large number of peptide hormones, a modification essential for the bioactivity of these peptides. 2. PAM activity was measured in membrane and soluble fractions of atrial and ventricular homogenates by monitoring the amidation of iodinated synthetic substrate ([125I]-Ac-Tyr-Val-Gly). 3. PAM activity in both species resided almost exclusively in the atria rather than the ventricles, and similar levels of activity were found in left and right atria. Membrane-associated activity was 50-to 100-fold greater than soluble activity in the sheep, yet was only five- to 10-fold greater in the rat, indicating a larger proportion of soluble enzyme in the rat atrium. 4. Similar apparent Km values were found for atrial membrane-associated activity in both species (15.6 and 17.4 mumol/L for rat right and left atria, 16.7 and 15.6 mumol/L for sheep right and left atria); however, the maximum velocity (Vmax) levels were higher in the rat (40.5 and 43.9 pmol/micrograms per h vs 12.8 and 15.1 pmol/micrograms per h). 5. Because expression of many peptides and processing enzymes can be regulated by steroid hormones, the possible effects of chronic glucocorticoid administration (1 mg dexamethasone i.m. twice daily for 10 days) on PAM levels were tested in four sheep, with four sheep receiving saline only as controls. There was no discernible effect of dexamethasone on either the distribution or the kinetics of PAM activity in the sheep heart.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1993

Monoclonal antibodies to arginine vasopressin receptor bind to liver, kidney and pituitary membranes.

1. A vasopressin binding protein purified from rat liver membranes was used to immunize Balb/c mice and, subsequently, for the screening of hybrids raised in two different cell fusions. 2. Three hybrids were obtained which secreted monoclonal antibodies (MoAb) that bound to the purified solubilized receptor as detected by an enzyme-linked immunosorbent assay technique. All three MoAb immunoprecipitated the purified receptor. 3. In addition, the MoAb bound in a concentration-dependent manner to crude liver, kidney and anterior pituitary membranes, tissues known to contain arginine vasopressin (AVP) receptors but not to cardiac ventricle membranes which lack AVP receptors. 4. However, the binding of [125I]-[d(CH2)5,Sar7]AVP (a specific radiolabelled V1 antagonist) to the membrane-bound receptor was not inhibited by these antibodies. 5. These results suggest that MoAb recognize epitopes which are common to rat liver, kidney and anterior pituitary membranes but are not at the ligand binding site.

Animals↗

PubMed1993

Ovine anterior pituitary proopiomelanocortin gene expression is not increased by ACTH secretagogues in vitro.

Recent reports have demonstrated that the secretion of ACTH from sheep anterior pituitary primary cultures is markedly stimulated by arginine vasopressin (AVP) but not by CRF, and that AVP-stimulated ACTH secretion is potentiated by CRF. It has also been reported that AVP increases total ACTH content (secreted plus intracellular ACTH), suggesting that AVP stimulates POMC biosynthesis in the ovine anterior pituitary. These observations differ from the rat, in which CRF is the most potent of the ACTH-releasing factors and the only ACTH secretagogue which stimulates POMC gene expression and biosynthesis. The second messenger pathways which mediate CRF- and AVP-stimulated ACTH release (protein kinase A and protein kinase C, respectively) are the same in sheep and rat corticotrophs. The present studies were undertaken to determine if ovine POMC gene expression, unlike the rat POMC gene, is stimulated by AVP via the protein kinase C pathway. A 295 base pair portion of the ovine POMC gene was isolated using polymerase chain reaction and sequenced. Ovine POMC messenger RNA (mRNA) levels were quantitated using this partial complementary DNA clone in a solution hybridization/nuclease protection assay with cytoplasmic RNA from sheep anterior pituitary primary cultures which had been treated with various combinations of ACTH secretagogues or with glucocorticoids for 18 h. Treatment with AVP, alone or with CRF, greatly increased total and secreted ACTH levels; however, the amount of POMC mRNA in these cells was not significantly increased. Treatments which stimulated secretion to a lesser extent and did not alter total ACTH levels (CRF alone, cAMP alone, or with phorbol ester) were associated with a decrease in POMC mRNA levels relative to untreated cells. Glucocorticoid treatment decreased both total ACTH and POMC mRNA levels. Taken together, the data demonstrate a lack of secretagogue-induced stimulation of POMC mRNA levels concomitant with increased total ACTH levels, an unexpected result given the close association between secretion of POMC-derived peptides and POMC gene expression in other mammalian corticotroph systems.

Adrenocorticotropic Hormone↗

PubMed1992

Distribution and characterization of peptidylglycine alpha-amidating monooxygenase activity in the ovine brain and hypothalamo-pituitary axis.

The production of alpha-amidated peptide hormones from their glycine-extended precursors is catalyzed by the specific enzyme peptidylglycine alpha-amidating monooxygenase (PAM). In the present study, the distribution and subcellular localization of PAM activity in the sheep brain was examined and compared with known sites of amidated peptide synthesis and release. Of the brain regions assayed, the preoptic anterior and medial basal areas of the hypothalamus contained the greatest concentration of amidating activity. Lower concentrations (greater than 3-fold less) were found in the anterior and neurointermediate pituitary, median eminence, cerebral cortex, hippocampus, pons-medulla, and brainstem. Very low amounts of activity were present in the cerebellum and pineal gland. In most tissues tested, PAM activity was 40-75% higher in the membrane-associated fraction than in the soluble fraction. In the hypothalamus, affinity constants were identical for both membrane-associated and soluble fractions, and ranged from 12.3-13.3 microM. Maximal velocity was higher in the membrane fraction (4.7-4.8 pmol/microgram.h) than in the soluble fraction (2.6-2.9 pmol/microgram/h). Levels of amidating activity in hypophysial-portal and jugular plasma were similar and were 20- to 25-fold lower than in hypothalamic extracts. Insulin-induced hypoglycemia did not significantly alter PAM levels in portal or peripheral plasma, suggesting that amidating activity is not released during this stress. These results indicate that the hypothalamus is the richest source of amidating activity in the sheep brain, and suggest that amidation of neurohypophysial and hypothalamic releasing peptides may occur before axonal transport, given the much lower levels in median eminence, neurointermediate pituitary, and portal plasma.

Amino Acid Sequence↗

PubMed1991

The interaction of a Huntington disease factor with receptors for the neurotoxin kainic acid.

A factor from mammalian and human brain, which inhibits the rate of migration of leukocytes obtained from sufferers from Huntington disease (Walls and Ruwoldt, 1984), inhibited the specific binding of the neurotoxin [3H]kainic acid to rat brain synaptic membranes. The factor was present in sucrose-particulate but not in soluble fractions from rat sub-cortical tissue, and was destroyed by tryptic digestion. Whereas an ammonium sulfate fraction of direct saline extracts of brain (Walls and Ruwoldt, 1984) gave poor chromatography on HPLC, prior separation of a sucrose-particulate fraction resulted in much improved chromatography. There was a good concordance between leukocyte migration inhibitory activity and [3H]kainic acid binding inhibitory activity. The factor may be an endogenous modulator of the kainic acid subset of receptors for the excitatory neurotransmitter glutamic acid.

Animals↗

PubMed1991

Identification of a non-covalent oxytocin/neurophysin-I complex in the bovine ovary.

Acid extracts of bovine preovulatory granulosa cells and corpora lutea (CL) were subjected to high-performance liquid chromatography (HPLC) and found to contain two peaks of immunoreactive (ir) oxytocin (OT), one corresponding to authentic OT and the second eluting 8 min later. The second peak was more abundant than authentic irOT in preovulatory follicles and in the early CL, but became less abundant as the CL matured (mid luteal) and was close to the limit of detection in the late CL. This peak could be detected only by an OT antiserum which recognized both the biologically active form of OT, as well as the post-translational processing intermediate Gly10-extended oxytocin. A second more specific OT antiserum (OT-933) did not recognize the second peak as strongly. Further analysis of the second peak revealed a complex of OT bound to its neurophysin (NP-I) which could be dissociated under denaturing conditions. Furthermore, we were able to create this complex in vitro by combining the two materials together under acid conditions, similar to the pH predicted in secretory granules, but not under neutral conditions. Measuring irNP-I by radioimmunoassay showed a single peak with a similar retention time to the OT/NP-I complex, confirming the identity of the unknown peak. Incubation of CL slices in culture showed a time-related release of both OT and NP-I, with OT having a greater rate of release in the mid luteal CL. These data suggest the presence of an OT/NP-I complex in the bovine preovulatory granulosa cells and CL, as well as the unbound peptide presumably within the secretory granules. The ratio of OT/NP-I complex and free peptide changes with ageing of the the CL, perhaps indicating regulated differences in the post-translational processing of the prohormone.

Animals↗

PubMed1990

Morphine decreases LH secretion in ovariectomized ewes only after steroid priming and not by direct pituitary action.

To determine whether opiates directly modulate pituitary LH secretion in vivo, morphine was administered to hypothalamo-pituitary-disconnected (HPD) ewes which were receiving exogenous pulses of GnRH. To define the steroidal background which is permissive to a morphine-induced decrease in LH secretion, ovariectomized (OVX) ewes were treated as follows in groups of four: group 1, no implant; group 2, small 17 beta-estradiol (E2) (1 cm long x 0.33 diameter) and progesterone (P) implants; group 3, medium E2 (1 cm long x 0.46 diameter) and P implants, and group 4, medium E2 implants. Jugular blood samples were taken at 10-min intervals for 9 h, during which there was a 3-hour pretreatment period, a 3-hour treatment period when the sheep were given six intravenous injections of 10 mg morphine every 30 min, and a 3-hour run-off period. Morphine inhibited the mean plasma concentrations of LH and LH pulse frequency in group 3 only, and in 2/4 ewes in this group LH secretion was abolished and did not return to a pulsatile mode during the 3-hour run-off sampling period. In a second experiment designed to test the pituitary action of morphine, OVX-HPD ewes were primed with medium E2 and P implants and were given hourly pulses of 250 ng GnRH intravenously. Jugular blood samples were taken around each GnRH pulse over an 8-hour period. The first three pulses served as a control sampling period, after which the sheep were treated with morphine (six intravenous injections of 10 mg morphine every 30 min).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

The methyltrienolone binding protein of human placenta requires nicotinamide adenine dinucleotide cofactor(s) for steroid binding.

The methyltrienolone binding protein (MTBP) found in human placental cytosol was found to require a low molecular weight modulator for steroid binding activity. Purification and characterization of the modulating activity showed that NAD+ is the endogenous substance responsible for activating MTBP to a form capable of steroid binding. The hierarchy of potency of exogenously added nucleotides is NADH greater than NAD+ = NADPH = NADP+. An investigation of the tissue distribution of human MTBP demonstrated that MTBP binding activity was present in placenta and chorion but absent from amnion and umbilical cord. Preliminary studies showed that rat, mouse, and rabbit placenta do not contain MTBP and suggest that MTBP may be a species-specific protein.

Amnion↗

PubMed1990

Arginine vasopressin and corticotropin releasing factor: binding to ovine anterior pituitary membranes.

In the sheep, in contrast to the rat, arginine vasopressin (AVP) is a more potent stimulus to ACTH secretion from the anterior pituitary (AP) than CRF. To further explore this difference, we have compared [3H]AVP and [125I]-[Nle21 Tyr32]ovine CRF binding in membranes prepared from rat and sheep AP. Between species, no difference in affinity of binding was found for either ligand. In contrast, the concentration of AVP receptors in sheep AP was twice that in rat, whereas that of CRF receptors was only one tenth. AVP receptor concentration in sheep AP was not altered by chronic (10 day) dexamethasone administration, but fell to 60% of control after chronic (60 day) hypothalamo-pituitary-disconnection. The increased level of AVP receptors and the much lower level of CRF receptors in sheep compared with rat may thus provide an explanation for our previous findings of increased sensitivity to AVP and a very poor response to CRF in stimulating ACTH release from the sheep AP. In addition the finding that AVP receptor numbers are reduced in the hypothalamo-pituitary-disconnected sheep suggests that hypothalamic factors may play a role in regulating AVP receptor concentration in the ovine AP gland.

Animals↗

PubMed1989

Gonadal steroids and anterior lobe dynorphin in the male rat.

Immunoreactive (ir)-dynorphin levels were measured, and the species characterized by high performance liquid chromatography (HPLC), in the pituitary and hypothalamus of intact and castrate male rats. On HPLC, ir-dynorphin co-eluted with authentic dynorphin A 1-8, dynorphin A 1-17 and dynorphin 1-32 in the hypothalamus and intermediate lobe; in two different reversed phase (RP)-HPLC systems, anterior lobe ir-dynorphin co-eluted uniquely with dynorphin 32 (4K dynorphin). Anterior lobe levels of total ir-dynorphin were significantly lowered 7 days after castration, while HPLC profiles in all tissues remained unchanged. The change in anterior pituitary ir-dynorphin levels was reversed in a dose-related manner by dihydrotestosterone (15-500 micrograms/100 g b. wt/day); estradiol benzoate (3 micrograms/100 g/day) was without effect. The changes on castration and androgen administration suggest that gonadal steroids play a role in the regulation of dynorphin, as well as gonadotrophins and prolactin, within the anterior pituitary gland.

Animals↗

PubMed1989

Immunocytochemical demonstration of mineralocorticoid receptors in rat and human kidney.

Using a polyclonal antiserum against the hinge region of the recently cloned human mineralocorticoid receptor (MR) and indirect peroxidase immunohistochemistry, we have shown MR-like immunoreactivity (LI) in superficial nephron segments, including distal convoluted tubule, connecting piece and initial cortical collecting duct. The absence of staining in cells tentatively identified as intercalated cells on light microscopy was confirmed by pre-embedding electron microscopy. Though the intracellular distribution of immunostaining varied with the fixative used, the cellular distribution of MR-LI is in good general agreement with earlier micropuncture and autoradiographic studies.

Animals↗

PubMed1989

Dopaminergic agents differentially regulate both processing and content of alpha-N-acetylated endorphin and alpha-MSH in the ovine pituitary intermediate lobe.

Hypothalamo-pituitary disconnection (HPD) in the sheep results in a two-fold increase in pituitary intermediate lobe (IL) immunoreactive (ir)-alpha-N-acetylated endorphin (NacEP) and ir-alpha-melanocyte-stimulating hormone (alpha MSH) content. The rise in IL NacEP content is accompanied by a markedly altered pattern of processing, in that NacEP1-27 becomes the dominant molecular species with a complementary fall in Nac alpha-EP and Nac gamma-EP. To determine if these effects reflect the loss of descending dopaminergic neuronal input to the IL, we have chronically treated two groups (n = 4 per group) of normal sheep with the dopamine antagonist haloperidol or the dopamine agonist bromocriptine; a group of HPD sheep were also treated with bromocriptine. Acid extracts of IL were diluted and ir-alpha-MSH and ir-NacEP content determined by radioimmunoassay; aliquots were submitted to reversed-phase HPLC and collected fractions similarly assayed. Bromocriptine lowered ir-NacEP and ir-alpha-MSH by about 30%; on HPLC the ir-NacEP profiles, and perhaps to a lesser extent those for ir-alpha-MSH were qualitatively similar to untreated controls. In contrast, haloperidol increased by about 45% both ir-NacEP and ir-alpha-MSH levels and produced a marked change in the ir-NacEP molecular profile, with NacEP1-27 becoming the predominant molecular form and other species representing only minor components in the chromatogram. In the treated HPD group, bromocriptine partially restored the processing profiles previously observed in HPD animals to those found in untreated intact animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Arginine vasopressin is a much more potent stimulus to ACTH release from ovine anterior pituitary cells than ovine corticotropin-releasing factor. 1. In vitro studies.

Cultured rat and ovine anterior pituitary cells were treated with a range of doses (0.01-1,000 nM) of arginine vasopressin (AVP) and ovine corticotropin-releasing factor (CRF), alone or in combination, and medium and cell content of immunoreactive (ir-)ACTH determined. In rat cells, a dose-response curve to CRF was obtained, with a threshold dose of 0.1 nM; AVP was much less effective alone, but augmented CRF responses when administered with CRF. In ovine pituitary cells AVP markedly stimulated ACTH release in a dose-dependent fashion, and with a threshold of 0.1 nM; in contrast, CRF increased ACTH release over basal only at doses greater than 100 nM. In combination, subthreshold doses of AVP potentiated rat pituitary cell responses to CRF; addition of 1 nM of AVP to varying doses of CRF was more effective in terms of ACTH release than addition of 1 nM of CRF to increasing doses of AVP. In contrast, in ovine cells the addition of 1 nM CRF to increasing doses of AVP elicited a larger ACTH response than the addition of 1 nM AVP to increasing doses of CRF. Dexamethasone pretreatment (5 nM) for 48 h significantly decreased CRF potentiation of AVP-stimulated ACTH release in ovine cells. These studies confirm that CRF is a more potent stimulus of ACTH release than AVP in the rat, and establish that in contrast AVP is a much more potent stimulus of ACTH secretion than CRF in isolated ovine pituitary cells.

Adrenocorticotropic Hormone↗

PubMed1989

Generation of Met-enkephalin Arg6Phe7 immunoreactivity by proteolytic cleavage of mammalian plasma precursors by pepsin.

A region-specific antiserum raised against the C-terminal heptapeptide of proenkephalin A (Met-enk Arg6Phe7) was used in RIA studies to show that rat, human, and ovine plasma contain substrates (mol wt, 68K) that yield nanomolar amounts of Met-enk Arg6Phe7 (ME-RF) after treatment with pepsin under acid conditions. This ovine plasma-derived immunoreactivity diluted in parallel to the ME-RF standard in RIA and chromatographed as two low mol wt species (approximately 1K) which were less hydrophobic than the standard on size exclusion and reverse phase chromatography. The pepsin-generated material displaced [3H]naloxone from rat brain binding sites; its potency was about 1000-fold that of ME-RF, assuming near 100% cross-reactivity with the antiserum. Taken together these observations suggest that the pepsin-generated material is of similar mol wt and amino acid sequence to ME-RF, but differs with respect to opiate-binding efficacy, and that the plasma precursor is distinct from proenkephalin in both size and processing sites.

Animals↗

PubMed1989

Immunolocalization of renal mineralocorticoid receptors with an antiserum against a peptide deduced from the complementary deoxyribonucleic acid sequence.

We have synthesized three peptides corresponding to putative antigenic regions in the immunogenic domain, hinge region, and carboxy-terminus of the protein. A rabbit immunized with a peptide derived from the hinge region of the receptor produced an antiserum which showed 50% displacement with 20 pg peptide at a final serum dilution of 1:35,000. When the antiserum was immunopurified and applied to sections of intact rat and human kidney it stained cells lining segments corresponding to distal tubule, connecting piece, and initial cortical collecting duct, consistent with the known sites of mineralocorticoid action. In both human (formaldehyde-fixed) and rat (Bouin's solution) there was ample evidence for both nuclear and cytoplasmic staining. The thymus, in which previously we have found [3H]aldosterone binding to be below detection limits, showed little or no staining. Western blot analyses demonstrated that the polyclonal antibody recognized an epitope of the expected molecular size. The availability of antibodies to the mineralocorticoid receptor should, thus, facilitate investigation of the steroid specificity-conferring mechanism which allows mineralocorticoids, but not glucocorticoids, access to the nonselective receptor in the kidney.

Amino Acid Sequence↗

PubMed1988

Mineralocorticoid action: target tissue specificity is enzyme, not receptor, mediated.

Mineralocorticoid receptors, both when in tissue extracts and when recombinant-derived, have equal affinity for the physiological mineralocorticoid aldosterone and for the glucocorticoids cortisol and corticosterone, which circulate at much higher concentrations than aldosterone. Such receptors are found in physiological mineralocorticoid target tissues (kidney, parotid, and colon) and in nontarget tissues such as hippocampus and heart. In mineralocorticoid target tissues the receptors are selective for aldosterone in vivo because of the presence of the enzyme 11 beta-hydroxy-steroid dehydrogenase, which converts cortisol and corticosterone, but not aldosterone, to their 11-keto analogs. These analogs cannot bind to mineralocorticoid receptors.

Adrenalectomy↗

PubMed1988

Right atrial dilatation increases inositol-(1,4,5)trisphosphate accumulation. Implications for the control of atrial natriuretic peptide release.

Stretching the right atrium of isolated perfused [3H]inositol-labelled rat hearts was shown to stimulate the phosphatidyl-inositol turnover pathway as demonstrated by the accumulation of [3H]inositol-(1,4,5)trisphosphate and its degradation products. Stimulation was detectable after 1 min with larger increases observed after 10 or 20 min. These findings demonstrate that the myocardium can respond to dilatation by an activation of the phosphatidylinositol turnover pathway. Such a mechanism has implications for the release of atrial natriuretic peptide following right atrial distention.

Animals↗

PubMed1988

Effect of myiasis and acute restraint stress on plasma levels of immunoreactive beta-endorphin, adrenocorticotrophin (ACTH) and cortisol in the sheep.

Cutaneous myiasis in sheep arising from the activity of Lucilia cuprina larvae can result in significant physiological changes in susceptible animals. The stress imposed on the pituitary-adrenal axis of the sheep in response to myiasis and acute restraint is the subject of this investigation. Merino wethers were exposed to handling restraint, and blood sampling, during examination for blowfly strike; where necessary, they were treated for cutaneous myiasis. Significant changes in the plasma concentrations of immunoreactive beta-endorphin (beta-EP), ACTH and cortisol were found in sheep with extensive myiasis, as compared with unstruck sheep or those with only localized myiasis. In five susceptible sheep with extensive cutaneous myiasis, mean plasma levels of beta-EP, ACTH and cortisol were 307 +/- 71 pg ml-1, 953 +/- 58 pg ml-1 and 232 +/- 46 nmol l-1 respectively, compared with 818 +/- 89 pg ml-1, 641 +/- 41 pg ml-1 and 107 +/- 17 nmol l-1 in six unstruck sheep handled similarly. Whereas significant increases in plasma ACTH and cortisol can result from pituitary-adrenal responses to acute emotional or surgical stress, and are usually accompanied by a concomitant release of beta-EP from the pituitary, the present findings indicate a marked reduction in beta-EP levels and a significant increase in ACTH and cortisol in sheep following blowfly strike and acute handling restraint. This result suggests that cutaneous myiasis in susceptible sheep can alter the pituitary-adrenal response to acute restraint stress, and this could occur either by an alteration of precursor processing in the pituitary or by the selective release of ACTH.

Adrenocorticotropic Hormone↗