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Biomedical subjects

A D Kenny

Publications and source records attributed to A D Kenny.

At least 19 recordsLinked to original sources
PubMed1994

Effect of reserpine pretreatment on avian erythrocyte carbonic anhydrase activation by isoproterenol.

We studied the action of beta-adrenergic agonists on Japanese quail erythrocyte carbonic anhydrase (CA) in vitro. Earlier we had reported that epinephrine increased CA activity by 14%; the present study focused on an attempt to increase the size of this response. Washed erythrocytes from reserpine-treated (1 mg/kg daily i.m. for 3 days) and control birds were incubated for 40 min in the presence of isoproterenol 10(-6) mol/l. The activity of CA expressed as Wilbur-Anderson units/mg hemoglobin was increased by as much as 42% in reserpine-treated birds over the control depending on the conditions. Addition of 10(-5) mol/l of the beta-adrenergic antagonist propranolol inhibited the isoproterenol-induced effect in nonreserpinized birds. We conclude that pretreatment with reserpine, which was accompanied by a fall in plasma catecholamine levels, particularly epinephrine levels, enhanced the activation of CA by isoproterenol.

Animals↗
PubMed1992

Determination of acetazolamide in human serum by enzymatic assay.

Carbonic anhydrase (CA) inhibitors, such as acetazolamide (AZ), formerly used as diuretics, still play a role in the treatment of glaucoma, epilepsy, and altitude sickness. There is now hard evidence from both in vitro and in vivo studies in animals that carbonic anhydrase plays a vital function in bone loss. Acetazolamide blocks bone resorption in these experimental models. We have postulated that acetazolamide has potential for the treatment of human conditions associated with bone loss. In preparation for a clinical trial of acetazolamide's effectiveness in this regard, we developed an enzymatic method for determining the total concentration of acetazolamide in human serum. Acetazolamide is stripped from binding to serum proteins by adding 10(-6) M salicylic acid and adjusting the pH to 2.5, followed by ultrafiltration through a membrane (10 kD cutoff). The latter permits the free acetazolamide to enter the filtrate but retains any carbonic anhydrase (31 kD) which may contaminate the serum from hemolysis. The carbonic anhydrase inhibitory activity in the filtrate, representing the acetazolamide, is determined in a carbonic anhydrase assay using acetazolamide as the standard. Recoveries of acetazolamide added to human serum ranged from 83% to 94% depending on the concentration. Precision, as judged by the coefficient of variation, was 10.5%.

Acetazolamide↗
PubMed1992

Carbonic anhydrase inhibition by flurbiprofen and related agents.

Flurbiprofen, a nonsteroidal anti-inflammatory drug (NSAID), reduces bone resorption in periodontal disease. This therapeutic effect has been attributed to inhibition of prostaglandin synthesis. In view of the importance of carbonic anhydrase (CA) in bone resorption, we examined the CA-inhibitory properties of flurbiprofen using bovine and human CA II and compared them with those of acetazolamide and two other NSAIDs, ibuprofen and indomethacin. Flurbiprofen inhibited both human and bovine erythrocyte CA II but to a much lesser degree than acetazolamide. Ibuprofen and indomethacin were much less active in inhibiting CA II than flurbiprofen.

Animals↗
PubMed1987

Role of carbonic anhydrase in bone resorption: effect of acetazolamide on basal and parathyroid hormone-induced bone metabolism.

The effects of the carbonic anhydrase inhibitor acetazolamide on basal and parathyroid hormone (PTH)-induced bone metabolism were studied to evaluate the manner in which acetazolamide inhibits bone resorption. Half-calvaria from 5 to 6-day-old mice were cultured using the following treatments: control; acetazolamide (10, 33, or 100 microM); PTH (16.7 nM bovine PTH 1-34); acetazolamide + PTH. The effects of acetazolamide on PTH-induced cAMP accumulation and protein synthesis were determined. Media from bones cultured for 48 hours were analyzed for calcium to assess bone resorption, glucose to assess calvarial glucose utilization, and lactic acid to assess calvarial lactic acid release. Media were also assayed for beta-glucuronidase activity as an indicator of lysosomal enzyme release and for lactate dehydrogenase activity as an indicator of cytosolic enzyme release and cytotoxicity. Acetazolamide at 100 microM completely inhibited PTH-induced bone resorption. This inhibition did not appear to be due to cell death, as acetazolamide did not increase lactate dehydrogenase release. Acetazolamide had no effect on PTH-enhanced cAMP levels, indicating that receptor binding and adenylate cyclase activation were unaffected. Acetazolamide alone did not alter calvarial protein synthesis, but did significantly inhibit protein synthesis in the presence of PTH. PTH significantly enhanced calvarial glucose utilization, lactic acid release, and beta-glucuronidase release. Acetazolamide inhibited all of these PTH-induced parameters in a manner that roughly paralleled its inhibition of bone resorption; acetazolamide alone had no effect on the basal values. Our results indicate that acetazolamide inhibition of bone resorption in vitro may involve general alterations in hormonally stimulated bone cell metabolism secondary to carbonic anhydrase inhibition.

Acetazolamide↗
PubMed1986

Vitamin D3 and avian bone in vitro: stimulation of calcium movement into Japanese quail calvaria.

Addition of 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) to cultured neonatal mouse calvaria has consistently led to bone resorption as determined by an increase in medium calcium. No such effect on avian bone has been widely reported. We have tested the in vitro effects of 1,25(OH)2D3 on 48- and 96-hour cultures of calvaria removed from both sexes of Japanese quail at various ages. No effect of 1,25(OH)2D3 on net calcium movement was seen in cultures of calvaria removed from neonatal (5-day), 1-week, and 2-week-old quail. In cultures of calvaria from 6-week-old female quail, addition of 1,25(OH)2D3 resulted in a fall in medium total and ionic calcium concentrations after 48 and 96 hours of incubation, indicating an uptake of calcium by the bones. A maximal effect was seen at 1 X 10(-7) M 1,25(OH)2D3. Bones removed from male and female quail showed no difference in response between the sexes. Bones removed from 5-, 4-, and 3-week-old male and female quail exhibited a progressively decreasing response with decreasing age. After 6 weeks of age female birds begin to exhibit hypercalcemia associated with reproductive activity. Bones removed from 7- and 8-week-old female quail responded to 1,25(OH)2D3 in a similar fashion whether they were hypercalcemic (7 weeks, 20.3 mg/dl; 8 weeks, 31.7 mg/dl), or normocalcemic (10.1 mg/dl). This stimulatory effect of 1,25(OH)2D3 on calcium uptake by avian bone is in sharp contrast to the hormone's effect in cultures of perinatal mammalian bones, which consistently respond to 1,25(OH)2D3 by releasing calcium into the medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Age Factors↗
PubMed1986

Vitamin D3 and avian bone in vitro: specificity of effect on Japanese quail calvaria.

Calcitriol (1,25(OH)2D3) has been shown, under certain conditions, to elicit an in vitro response in adult avian calvarium which may be interpreted as calcium uptake by the bone. The present investigation was undertaken to study the specificity of this response. Calvaria were removed from 6-week-old female Japanese quail and cultured for periods of up to 96 hours at 37 degrees C in 5% CO2/95% air. 1,25(OH)2D3 induced a fall in the medium total and ionic calcium concentrations at both 48 hours and 96 hours of incubation; these responses were not blocked by the presence of 10(-4) M acetazolamide. Bovine parathyroid hormone (bPTH(1-34] at 10(-7) M, and dibutyryl cyclic AMP (DBcAMP) at 10(-4) M, had no effect on the medium calcium. In contrast, forskolin at 10(-4) M induced a marked fall in medium calcium concentrations, particularly at 48 hours. The specificity was also studied with respect to vitamin D3 and its two major metabolites. 1,25(OH)2D3 exhibited a bell-shaped dose-response relationship with the maximal effect at 10(-7) M. In contrast, the other two compounds elicited no effects at 10(-7) M or 10(-6) M; significant responses were observed at 10(-5) M with both agents. In general, 25-dihydroxyvitamin D3 (25OHD3) was more potent than vitamin D3. These findings suggest that the medium calcium response to 1,25(OH)2D3, interpreted as calcium uptake by the cultured adult avian bone, is relatively specific among calcemic agents; the response was elicited by forskolin but not by bPTH(1-34) or DBcAMP.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetazolamide↗
PubMed1986

Cyclic AMP and the vascular action of parathyroid hormone.

The involvement of tissue cAMP in the vasodilating action of parathyroid hormone (PTH) was investigated. The bovine active fragment bPTH-(1-34) was used in all studies. In anesthetized dogs, theophylline, a phosphodiesterase inhibitor, potentiated the hypotensive action of bPTH-(1-34) at the dose of 1 microgram/kg. The potentiation was related to the dose of theophylline infused. In an in vitro rat tail artery helical strip assay, dibutyryl cAMP produced dose-related relaxation in arginine vasopressin (AVP) constricted blood vessels. bPTH-(1-34) also produced dose-related relaxation in the tail artery constricted by AVP. In the presence of isobutylmethylxanthine, another phosphodiesterase inhibitor, the bPTH-(1-34) dose--response curve was shifted to the left, indicating potentiation. Imidazole, which has phosphodiesterase stimulating activity, significantly decreased the in vitro vasorelaxing effect of bPTH-(1-34). In addition, bPTH-(1-34) increased significantly the rat tail artery cAMP content. b-PTH-(1-34) oxidized with hydrogen peroxide lost its vasorelaxing activity and was also ineffective in increasing the tail artery cAMP content. All these data strongly suggest that cAMP may be involved in eliciting the vasorelaxing action of bPTH-(1-34).

1-Methyl-3-isobutylxanthine↗
PubMed1986

Forskolin-induced bone resorption in neonatal mouse calvaria in vitro.

The role of cyclic adenosine 3',5'-monophosphate (cAMP) in inducing bone resorption was studied in neonatal mouse calvaria in vitro. Forskolin, a stimulator of adenylate cyclase, increased the medium calcium concentration at 96 hr of incubation, indicating enhanced bone resorption. Bone resorption was observed between 1 X 10(-4) and 1 X 10(-6) M forskolin; the maximal effect was at 1 X 10(-5) M and there was no effect at 1 X 10(-7) M. Lactic acid release was increased during the 96 hr of incubation in proportion to the calcium release in the media. The bone acid phosphatase activity was increased and the alkaline phosphatase activity was decreased. Bone carbonic anhydrase activity was increased more than twofold. Forskolin-induced bone resorption was significantly but incompletely inhibited by 10(-4) M acetazolamide, a carbonic acid anhydrase inhibitor. These findings support the concept that carbonic anhydrase plays a significant role in bone resorption.

Acetazolamide↗
PubMed1986

Bone resorption induced by parathyroid hormone and dibutyryl cyclic AMP: role of carbonic anhydrase.

The role of carbonic anhydrase in bone resorption induced by parathyroid hormone (PTH) and dibutyryl cyclic AMP (DBcAMP) was studied using an in vitro neonatal mouse half-calvarial culture system. Both PTH (16.7 nM) and DBcAMP (0.3 mM) were effective in stimulating bone resorption, as assessed by measuring changes in media calcium concentrations. Bones treated with PTH or DBcAMP for 96 hr contained significantly greater carbonic anhydrase activity than control bones [PTH Treated/Control (T/C) = 2.44; DBcAMP T/C = 2.34]. Both PTH and DBcAMP significantly enhanced calvarial acid phosphatase activity relative to control values [PTH T/C = 1.48; DBcAMP T/C = 1.30]. Neither PTH nor DBcAMP significantly altered calvarial alkaline phosphatase activity. Bone resorption induced by PTH and DBcAMP was inhibited by the carbonic anhydrase inhibitor acetazolamide, but not by the acetazolamide analog CL 13,850 (N-t-butylacetazolamide), which does not inhibit carbonic anhydrase. These results support the concepts that PTH-induced bone resorption requires the action of osteoclastic carbonic anhydrase and that the action of PTH on bone is mediated, in part, by the action of cyclic AMP.

Acetazolamide↗
PubMed1985

Role of carbonic anhydrase in bone: partial inhibition of disuse atrophy of bone by parenteral acetazolamide.

Carbonic anhydrase inhibitor acetazolamide blocks the hypercalcemic response to parathyroid hormone (PTH) and to dibutyryl 3',5'-cyclic AMP in the nephrectomized-parathyroidectomized rat. In addition, we have reported that acetazolamide, when incorporated in the diet, partially prevents denervation-induced bone loss in a rat model of disuse osteoporosis. The present study compares the effectiveness of orally and subcutaneously administered acetazolamide in preventing denervation-induced bone loss in the rat model. The rats were treated with acetazolamide either orally, by incorporation in the diet of concentrations of 0.2, 0.5, or 1.5% for 15 days, or parenterally by two different subcutaneous methods of administration. The latter included either injection twice daily for 15 days or continuous infusion for 8 days using an osmotic minipump. It was found that parenteral administration was as effective in partially preventing denervation-induced bone mass changes as oral administration. In addition, protection by the parenteral route could be accomplished with much smaller daily doses; continuous infusion required the least daily dose. Approximately 50% protection was observed to occur with daily doses of 1,094, 129, and 8 mg/kg body weight for the oral, subcutaneous injection, and subcutaneous infusion routes respectively. These findings are consistent with our concept that carbonic anhydrase plays a significant role in bone metabolism.

Acetazolamide↗
PubMed1985

Role of carbonic anhydrase in bone resorption induced by 1,25 dihydroxyvitamin D3 in vitro.

The present investigation was undertaken to study the role of carbonic anhydrase in 1,25 dihydroxyvitamin D3-induced bone resorption. Calvaria were removed from 5- to 6-day-old mice and cultured for periods up to 96 h in Dulbecco's Modified Eagle Medium (high glucose, 4,500 mg/dl) supplemented with antibiotics and either heat-inactivated horse and fetal calf sera or bovine serum albumin. The experimental cultures contained 1 X 10(-8) M 1,25 dihydroxyvitamin D3 (1,25(OH)2D3). All cultures were incubated at 37 degrees C in 5% CO2/95% air. Bone resorption was assessed by release of stable calcium into the medium. Bone enzymes (acid and alkaline phosphatases and carbonic anhydrase) were determined following homogenization in 0.25 M sucrose. The effects of 1,25(OH)2D3 were studied in the presence and absence of the carbonic anhydrase inhibitor acetazolamide and its analogue (CL 13,850), which lacks inhibitory activity. Acetazolamide inhibited 1,25(OH)2D3-induced calcium release in a dose-dependent fashion from 10(-5)-10(-4)M. When added to the cultures at a concentration of 1 X 10(-4)M, acetazolamide completely blocked the 1,25(OH)2D3-induced calcium release, a phenomenon not seen with an equimolar concentration of CL 13,850. The most significant finding was that 1,25(OH)2D3-induced calcium release was accompanied by a significant increase in the carbonic anhydrase activity of bone at both 48 (treated/control ratio = 2.05) and 96 (treated/control ratio = 2.59) hours.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetazolamide↗
PubMed1985

Role of carbonic anhydrase in bone resorption induced by prostaglandin E2 in vitro.

The possible role of carbonic anhydrase in bone resorption induced by prostaglandin E2 (PGE2) was studied using an in vitro neonatal mouse calvarial culture system. PGE2 (10(-6) M) was effective in stimulating resorption, as assessed by calcium release into culture media. This enhanced resorption was accompanied by significant increases in calvarial carbonic anhydrase activity over control values at 48 and 96 h. At 48 h, bones treated with PGE2 had 20% more carbonic anhydrase activity than controls. By 96 h, treated bones contained 79% more carbonic anhydrase activity than controls. PGE2-induced bone resorption was inhibited by the carbonic anhydrase inhibitor acetazolamide in a dose-dependent fashion from 10(-5) to 10(-4) M, with 77% inhibition observed at 10(-4) M. The acetazolamide analogue CL 13,850 (N-t-butylacetazolamide), which does not inhibit carbonic anhydrase, failed to inhibit PGE2-induced resorption. These results are consistent with the hypothesis that carbonic anhydrase is a necessary component of the osteoclastic bone resorptive mechanism.

Animals↗
PubMed1985

Role of carbonic anhydrase in bone: plasma acetazolamide concentrations associated with inhibition of bone loss.

Earlier reports from our laboratory have indicated that the carbonic anhydrase inhibitor acetazolamide blocks the hypercalcemic response to parathyroid hormone. In addition, we have reported that acetazolamide when administered by several routes partially prevents denervation-induced bone loss in a rat model of disuse osteoporosis. Continuous subcutaneous infusion required the least daily dose (8 mg/kg). The present study extends these earlier findings in several ways. It was found that in partially preventing denervation-induced bone loss: (1) incorporation of 1 M THAM [tris(hydroxymethyl)aminoethane] enhanced the potency of acetazolamide such that it was effective at daily doses of 0.6 mg/kg; (2) acetazolamide in the presence of 1 M THAM was effective at plasma concentrations as low as 50 ng/ml which are more than 500-fold less than peak plasma concentrations normally encountered in the human when acetazolamide is being used as a therapeutic agent; and (3) another inhibitor, benzolamide, was also effective by continuous subcutaneous infusion.

Acetazolamide↗
PubMed1985

Activation of avian medullary bone osteoclasts by oxidized synthetic parathyroid hormone (1-34).

Synthetic bovine parathyroid hormone (1-34) [bPTH(1-34)] has been treated with hydrogen peroxide and assayed for the effect of such treatment on the ability of bPTH(1-34) to activate medullary bone osteoclasts during their quiescent period in the early phase of the ovulatory cycle in Japanese quail. In addition, the same batches of oxidized and unoxidized bPTH(1-34) were assayed for their hypercalcemic activity in Japanese quail and their capacity to stimulate renal adenylate cyclase activity in the same species. Three groups, each consisting of five 5-month-old egg-laying Japanese quail (Coturnix coturnix japonica), were used. Between 4 to 5 hr after oviposition the three groups were injected intraperitoneally with acid saline (control) solution, bPTH(1-34) at 40 micrograms/bird, or oxidized bPTH(1-34) at 40 micrograms/bird, respectively. Twenty minutes after injection, the femoral bones were removed, split, fixed, and appropriately processed for examination by electron microscopy. Both oxidized and unoxidized bPTH(1-34) stimulated the development of osteoclast ruffled borders within 20 min after injection of the hormone preparations. As anticipated from previously published work from this laboratory, oxidized bPTH(1-34) retained its hypercalcemic activity and lost its capacity to stimulate renal adenylate cyclase activity in the Japanese quail. These results support, but do not prove, the contention that bPTH(1-34) exhibits its responses in the Japanese quail through the mediation of more than one type of receptor.

Adenylyl Cyclases↗
PubMed1985

Survey of vitamin D metabolite levels during growth and development in Japanese quail.

Plasma calcium and vitamin D metabolite levels were monitored during growth and development in male and female Japanese quail. In male Japanese quail, plasma calcium levels were constant (range 9.0 to 10.9 mg/dl); the 1,25-dihydroxyvitamin D [1,25-(OH)2D] levels remained undetectable in all age groups. Plasma calcium levels in the females rose at 6 weeks of age and remained elevated. The female 1,25-(OH)2D levels increased from undetectable levels at 3 to 5 weeks to a peak of 297 pg/ml at 8 weeks and remained elevated at 12 weeks of age in ovulating birds. Twelve-week old female without an egg in their oviduct had plasma 1,25-(OH)2D levels similar to males and immature females. No marked changed occurred in plasma 25-(OH)D levels due to sex or age. Plasma 24, 25-(OH)2D levels stayed relatively constant at about 2 ng/ml for both male and females at all ages studied.

24,25-Dihydroxyvitamin D 3↗
PubMed1984

Cardiac actions and structural--activity relationship of parathyroid hormone on isolated frog atrium.

The hypercalcemic and hypotensive actions of parathyroid hormones (PTH), parathyroid extract, and synthetic PTH-(1-34) have been reported in many different animals. However, their cardiac action was only recognized recently. In the present study, experiments were designed to examine (1) whether PTH possesses any cardiac action on the isolated frog atrium, and (2) the importance of methionine and arginine of bPTH-(1-34) in cardiac action. Six different bPTH analogs were tested in frog atria in vitro: bPTH-(1-34); H2O2-treated bPTH-(1-34); [Nle8, Nle18, Tyr34]-bPTH-(1-34); H2O2-treated [Nle8, Nle18, Tyr34]-bPTH-(1-34); arginine-modified bPTH-(1-34); and arginine-regenerated bPTH-(1-34) (Nle-norleucine). bPTH-(1-34) produced positive chronotropic and inotropic responses. Oxidation with hydrogen peroxide abolished both actions of bPTH-(1-34). [Nle8, Nle18, Tyr34]-bPTH-(1-34) also possesses this cardiac-stimulating property. The H2O2 treatment of this analog did not alter the chronotropic action, but a decrease in the inotropic action was observed. The arginine-modified bPTH-(1-34) was much less potent than its native hormone, while the reversal of the arginine modification partially restored the biopotency. From these data, it is concluded that (1) bPTH-(1-34) possesses both positive chronotropic and inotropic effects on the frog atrium, (2) the cardiac actions of bPTH-(1-34) may be closely related with its hypotensive property, (3) methionines are not necessary for the cardiac actions, and (4) arginines are important for the cardiac-stimulating effects of bPTH.

Animals↗
PubMed1984

Uterine relaxing action of parathyroid hormone: effect of oxidation and methionine substitution.

The effects of bPTH-(1-34), oxidized bPTH-(1-34),[Nle8,Nle18, Tyr34] bPTH-(1-34) amide, and oxidized [Nle8,Nle18,Tyr34]bPTH-(1-34)amide were tested in an in vitro rat uterine assay. When bPTH-(1-34) was treated with hydrogen peroxide (H2O2), the ability of this peptide to reduce oxytocin-stimulated uterine contraction in vitro was no longer evident. An analogue of bPTH-(1-34), in which the methionines at positions 8 and 18 were replaced with norleucine ([Nle8,Nle18,Tyr34]bPTH-(1-34)amide), was capable of reducing oxytocin-stimulated uterine contraction. However, when the [Nle8,Nle18,Tyr34]bPTH-(1-34)amide was oxidized, it retained the ability to reduce uterine contraction. Since we have previously shown that H2O2 oxidation affected only the methionines, these results suggest that the methionines are not necessary for the uterine activity of bPTH-(1-34). We have previously shown that oxidation of bPTH-(1-34) also destroys its blood vessel relaxing activity but has no effect in the rat or the Japanese quail hypercalcemic assays. These data combined with the results of the present studies suggest that the uterine and vascular smooth muscle relaxing properties of bPTH-(1-34) may require the same structural conformation and that this conformation is different from that required for the hypercalcemic action of the peptide.

Animals↗