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Biomedical subjects

K Purvis

Publications and source records attributed to K Purvis.

At least 91 records · Page 5Linked to original sources
PubMed1982

Rat sperm enzymes during epididymal transit.

The activities of cAMP and cGMP phosphodiesterases (EC 3.1.4.1), adenylate cyclase (EC 4.6.1.1) and protein carboxyl-methylase (EC 2.1.1.24) were measured in the particulate and soluble (105 000 g supernatant) fractions of washed spermatozoa isolated from five segments of the adult rat epididymis. The activities of both phosphodiesterases decreased during epididymal transit, whereas adenylate cyclase and protein carboxyl-methylase underwent a progressive increase, the latter showing the most marked alteration. Both cAMP and cGMP phosphodiesterases as well as the adenylate cyclase were all associated primarily with the particulate fraction, and the extent to which these enzymes were associated with the membranes increased as the spermatozoa passed through the epididymis. Sperm protein carboxyl-methylase activity was, on the other hand, predominantly soluble in all segments of the epididymis. Adenylate cyclase, cAMP phosphodiesterase and protein carboxyl-methylase activities were found predominantly in the sperm tails, whereas cGMP phosphodiesterase was equally distributed between heads and tails. These observations imply that the acknowledged increase in intracellular cAMP levels which occurs in spermatozoa during epididymal transit may be a consequence of both increased synthesis (adenylate cyclase) and reduced hydrolysis (phosphodiesterase).

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1982

Desensitization of FSH-responsive adenylyl cyclase in cultured immature Sertoli cells by homologous hormone.

Sertoli cell monolayers were prepared from 19-day-old rat testes. On day 7 of culture cells were incubated for 24 hr in the presence or absence of ovine follicle stimulating hormone (oFSH). Cells were harvested, and adenylyl cyclase responses of the membrane particles to FSH, human chorionic gonadotropin (hCG), isoproterenol, and fluoride (F-) were examined in the presence of either GTP or the nonhydrolyzable guanylyl nucleotide GMP-P(NH)P. Culturing the cells in presence of FSH caused a hormone specific desensitization of FSH-responsive adenylyl cyclase, whereas responses to isoproterenol and fluoride were unaffected. Activation of Sertoli cell adenylyl cyclase by GTP and GMP-P(NH)P showed no difference between cells preincubated with or without FSH, indicating that FSH did not change the activity of the G/F (or N) component or its interaction with the catalytic subunit of the adenylyl cyclase. FSH-responsive adenylyl cyclase in cultured Sertoli cells has been shown to be selectively desensitized by homologous hormone. The mechanism may involve alteration or loss of the FSH receptor or changes in the "coupling" of the FSH receptors to the G/F component of the adenylyl cyclase, since there was no alteration in the guanylyl nucleotide and fluoride activation.

Adenylyl Cyclases↗
PubMed1981

Cellular localization of the Mn2+-dependent adenylyl cyclase (AC) in rat testis.

In the present study we have examined the exact cellular localization of the Mn2+-dependent adenylyl cyclase (AC) in the rat testis. Several pieces of evidence indicate that this testis specific enzyme is exclusively localized to haploid cells in the testis: firstly, the appearance of Mn2+-dependent adenylyl cyclase coincides in time with the occurrence of haploid cells (IC) in the rat testis. Secondly, tests of tfm rats and mice, in which little or no haploid cells are found, exhibit negligible Mn2+-dependent AC activity. Finally, separation of various testicular cells by BSA gradient sedimentation (Staput fractionation), revealed that this enzyme activity follows the location of haploid cells; the highest specific AC activities being found in the fractions containing elongated spermatids. Some activity found in the primary spermatocyte fraction (4C cells) can to a large extent be explained by the contamination of haploid cells in this fraction.

Adenylyl Cyclases↗
PubMed1981

Effects of castration, sex steroids, LHRH and glucocorticoids on LHRH binding in the anterior pituitary of male rats.

In the present study we have examined the effects of gonadotrophin releasing hormone (LHRH), sex steroids and glucocorticoids on the binding of LHRH to receptors in the pituitary of male intact and castrated rats. In intact rats, LHRH (10 microgram/day) treatment for 11 days caused a significant increase in LHRH binding, whereas testosterone (500 microgram/day) or oestradiol (50 microgram/day) were inhibitory. 17-hydroxyprogesterone and dexamethasone were without effects. In castrated rats, LHRH caused a marginal decrease in LHRH binding. Much greater inhibition was observed with testosterone and oestradiol. 17-hydroxyprogesterone reduced binding to that of intact controls, whereas dexamethasone was ineffective. When different doses of sex steroids were tested, both oestradiol, testosterone and 5 alpha-dihydrotestosterone inhibited LHRH in a dose-dependent manner. The lowest doses of steroids causing significant inhibition of LHRH binding in castrated animals were 0.5, 50 and 500 microgram/day for oestradiol, 5 alpha-dihydrotestosterone and testosterone, respectively. The present study shows that pituitary receptors for LHRH are regulated both by sex steroids and LHRH itself.

Animals↗
PubMed1981

Age-related changes in [125I]hLH specific binding to rat interstitial cells.

Enriched Leydig cell suspensions were prepared from rats ranging from 5 to 80 days of age. The cells were incubated with [125I]hLH in vitro, and the proportion of LH binding cells determined by means of autoradiography. The proportion of 3 beta-hydroxy-steroid-dehydrogenase (3 beta-HSD) positive cells was also determined. By means of Scatchard analysis and determination of grain counts of LH positive cells, an increase in LH receptors per LH binding cells with increasing age was found. LH binding cells from animals 5 and 10 days of age had few LH receptors, more than a doubling occurred between day 10 and day 20, a significant increase was seen between day 20 and 40 whereas only a slight increase in LH receptors per LH binding cell was seen thereafter. Scatchard analysis and grain count analysis of labelled cells gave essentially the same results. The grain count distribution showed cells distributed around low grain values in animals of all ages, whereas a subpopulation of cells with high grain counts appeared in maturing and mature animals. Only a small fraction of LH positive cells had detectable levels of 3 beta-HSD activity in animals 5 and 10 days of age. With increasing age the proportion of 3 beta-HSD positive cells approached that of LH binding cells, indicating that rat interstitial cells acquire LH receptors well before any 3 beta-HSD activity can be traced.

3-Hydroxysteroid Dehydrogenases↗
PubMed1981

Temporal relationship between hCG induced desensitization of LH/hCG responsive adenylyl cyclase and downregulation of LH/hCG receptors in the rat testis.

A single s.c. injection of 75 IU of human chorionic gonadotropin (hCG) into adult male rats caused a transient desensitization of the LH/hCG responsive adenylyl cyclase (AC) in membrane particles from rat testis. Two hours after the injection of hCG, LH/hCG responsive AC was reduced by 40%, whereas LH binding was still normal. After 24 hr LH/HCG responsive AC was lost, whereas LH binding only showed a marginal decrease. During the next 2-3 days there was a gradual loss of LH/HCG receptors, which reached a nadir at day 4 (14% of control levels). At day y and 9 following the hCG injection LH/hCG receptors and LH/hCG responsive AC gradually returned towards normal. Fluoride (F-) stimulated AC activity showed only minor changes throughout the period investigated. The initial loss of AC responsiveness to hCG was associated with maximal levels of circulating hCG. However, desensitization persisted for several days after hCG was cleared form the circulation. AC desensitization preceded LH receptor downregulation and may support the notion that uncoupling of the catalytic subunit of the AC from Leydig cell LH/hCG receptors is a requirement for subsequent receptor internalization.

Adenylyl Cyclases↗
PubMed1981

Testicular cyclic nucleotide phosphodiesterase in the rat. Kinetic properties and changes with age.

The assay for cyclic nucleotide phosphodiesterase has been applied, with certain modifications, to the measurement of the soluble forms of these enzymes in the rat testis. The homogenization and incubation conditions were adjusted to achieve linear product formation as a function of time and protein concentrations and the resulting products were isolated by ion exchange chromatography using 5 mM HCl as the eluting agent. Phosphodiesterase activities were present in the testicular cytosol (105,000 g supernatant) of adult rats which were capable of hydrolyzing cAMP with a high (Km2 microM) and low Km20 microM) affinity and GMP with a relatively high affinity (Km3 microM). The low affinity cAMP enzyme activity could be stimulated with divalent ions such as calcium, magnesium, and manganese. At 18 days of age, all three enzyme activities were present in the testis, although both the high and low affinity cAMP phosphodiesterase displayed maximal rates (Vmax) that were only one third of the adult testis (when expressed per mg protein).

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1980

Changes of pituitary hormones in brain death.

In six patients with clinical and electroencephalographic signs of brain death, pituitary hormones such as prolactin, human growth hormone (GH), luteinizing hormone (LH), and thyrotrophin (TSH) were measured in blood close to the demonstration of intracranial circulatory arrest by angiography. In addition, pituitary hormone releasing tests and an insulin test were carried out in two patients. The results showed that no patient had a general decrease in hormone levels, according to their biological half life times, which suggests there still was some function in the hypothalamus and pituitary. This was supported by the results of the stimulation tests. It is concluded that in brain death some basal parts of the brain may still be perfused despite the fact that angiography indicates circulatory arrest in these areas.

Adolescent↗
PubMed1980

PGE1 and hCG responsive adenylyl cyclases in interstitial cells from rat testis: biphasic effects of calcium.

The effects of PGE1 (10 micrograms/ml), hCG (10 mIU/ml) and Ca2+ (0-10 mM) on rat interstitial cell adenylyl cyclase (AC) activity have been investigated. Like hCG, PGE2 also stimulated membrane bound AC, and both hormonal effects proved to be influenced by Ca2+. Low concentrations of Ca2+ (0.3 mM) enhanced hormonal stimulation, while high concentrations (up to 10 mM) suppressed AC activity. Relative stimulations caused by PGE1 and hCG were maximal in the presence of 0.6 mM Ca2+. Ovine FSH (NIH-S12), at concentrations causing maximal activation of AC in seminiferous tubules (12.5 micrograms/ml), had no effect on AC in interstitial cells. Germinal cell membranes displayed insignificant AC activity. We therefore conclude that the AC activity stimulable by PGE1 is located on Leydig cell membranes and that both PGE1 and hCG activities are susceptible to regulation by Ca2+.

Adenylyl Cyclases↗
PubMed1980

LH/hCG responsive adenylyl cyclase in rat testis and testes from testicular feminized male (Tfm) rats and mice; desensitization by homologous hormone.

Testes of testicular feminized (tfm) rats and mice, as well as of normal male rats contain an LH/hCG responsive adenylyl cyclase. Basal, as well as hCG stimulated activities were higher in tfm rats and mice than in normal rats. The presence of an LH/hCG responsive adenylyl cyclase in the testis of tfm rats and mice shows that the greatly elevated LH levels present in males having this syndrome, giving 80-90% reduction in LH/hCG receptors, do not cause an uncoupling of the remaining receptors from the adenylyl cyclase. It also shows that androgens are not essential for coupling of the LH/hCG receptors to the adenylyl cyclase. Injection of 200 IU of hCG into adult normal rats and tfm rats caused, after 48 h, a complete loss of LH/hCG stimulated adenylyl cyclase whereas the FSH responsive adenylyl cyclase in both animal preparations was maintained. Desensitization of the LH responsive adenylyl cyclase by hCG in normal rats, confirms previous studies showing lack of hCG stimulated cyclic-AMP secretion after a comparable dose of hCG in vivo. Similarly, hCG (50 IU) caused a transient loss of LH/hCG responsive adenylyl cyclase in tfm mice, with a complete disappearance of response after 24 h. At 48 and 72 h after injection of hCG the response gradually returned to normal. The fact that hCG caused a complete desensitization of the LH/hCG responsive adenylyl cyclase in both tfm rats and mice, proves that androgen receptor mediated events are not involved in hCG desensitization of the adenylyl cyclase in Leydig cells.

Adenylyl Cyclases↗
PubMed1980

Calmodulin regulation of testicular cyclic nucleotide phosphodiesterases.

Three different, calmodulin-dependent, isoenzymes of cyclic nucleotide phosphodiesterase (PDE) can be isolated by DEAE cellulose chromatography from the immature rat testis and shown to have different substrate specificities and kinetic behaviour. Calmodulin is regulating the activity of these enzymes by altering the maximal velocity (Vmax) rather than the Km of the reaction. Two of the 3 enzymes appear to be testis-specific, while the remaining possesses properties similar to that described for the calmodulin-dependent PDE found in other tissue.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1980

Cofactor dependency of soluble 3 alpha-hydroxysteroid oxidoreductases in rat testis, prostate, and epididymis.

The present paper demonstrates the presence of at least two 3-hydroxysteroid oxidoreductases (3-HSO) in rat testis, prostate, and epididymis cytosol preparations. The enzymes were either NADH or NDAPH dependent. Further investigation by Sephadex G-200 chromatogrphy revealed the presence of enzyme activities in the void volume (peak 1) and also eluting close to the methyl-carbonic anhydrase standard (mol wt, 34,000; peak 2). Enzyme activity in peak 1 was predominantly stimulated by NADH and that in peak 2 was stimulated mainly by NADPH. Both 3 alpha- and 3 beta-HSO activities were observed in testicular eluates from 1; 3 beta-Adiol accounted for up to 50% of the 5 alpha-androstanediols formed. In peak 2,3 alpha-HSO constituted more than 90% of the enzyme activity. In contrast, the prostatic and epididymal eluates revealed only 3 alpha-HSO activity; 3 beta-Adiol constituted less than 5% of the 5 alpha-androstanediols formed in either peak 1 or 2. The apparent Km values for enzyme activation reveal differences in sensitivity to cofactors for enzymes in peaks 1 and 2 and also among testis, epididymis, and prostate. NADH caused a very similar activation of enzyme activity in peak 1 or the prostate and epididymis (Km 50-100 micro M), whereas the enzyme in the testis was activated by much lower cofactor concentration (Km approximately 5 microM). It is possible that this enzyme activity may represent microsomal contamination. The enzyme activity in peak 2 revealed very similar sensitivity to NADPH in all three organs (Km, 0.6-1.7 microM), confirming previous studies from our laboratory that the soluble. NADPH-dependent enzymes in all three tissues are very similar, if not identical.

3-Hydroxysteroid Dehydrogenases↗