摘要:

We have investigated the hypoglycemic effects of the newly synthesized short-acting nonsulphonylurea hypoglycemic agent calcium (2S)-2-benzyl-3-(cis-hexahydro-2-isoindolinyicarbonyl)-propionate dihydrate (KAD-1229) in non-insulin-dependent diabetes mellitus (NIDDM) rats. NIDDM rats that were given a neonatal injection of 60 mg/kg streptozotocin showed a dose-dependent but attenuated response to oral administration of KAD-1229 and gliclazide, and their impaired glucose tolerance was improved but not normalized. We next produced, using a neonatal injection of 30 mg/kg streptozotocin, a mild type of NIDDM rat with less impaired glucose tolerance. These rats responded well to these insulinotropic hypoglycemic agents. Their impaired glucose and meal tolerance were completely normalized by oral administration of 3 mg/kg KAD-1229. The efficacy of KAD-1229 in this NIDDM rat model 1–3 h after oral glucose administration was comparable with similar doses of gliclazide, despite its shorter hypoglycemic action (compared with gliclazide), in fasting normal rats. In meal tolerance tests (20 kcal/kg; 1 cal = 4.2 J), KAD-1229 reduced abnormally enhanced plasma glucose levels 1–3 h after administration. This effect disappeared by 5 h. In contrast, gliclazide showed sustained hypoglycemic effects until 5 h after oral administration, with a lower postprandial (0.5–1 h) effect. These data indicated that the rapid- and short-acting efficacy of KAD-1229 would be beneficial and sufficient to control postprandial plasma glucose in NIDDM rats.Key words: KAD-1229, hypoglycemic agent, streptozotocin-induced non-insulin-dependent diabetes mellitus rats.

ISSN:0008-4212    

DOI:10.1139/y95-001

出版商:NRC Research Press    

年代:1995

数据来源: NRC

摘要:

The aromatic hydrocarbon receptor (AHR) is a soluble intracellular protein that mediates most, if not all, the toxic effects of polycyclic aromatic hydrocarbons, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 3-methylcholanthrene. Initial binding of specific AHR ligands occurs in the cytoplasm; after a "transformation" step the ligand∙receptor complex translocates to the cell nucleus and binds to specific DNA sequences, which act as transcriptional enhancers. We used a synthetic peptide – KLH conjugate corresponding to a 20 amino acid sequence at the N-terminal of the AHR to generate rabbit polyclonal anti-AHR antibodies. The antiserum was affinity purified, using the synthetic peptide conjugated to ovalbumin, and screened by western blot analyses, using [3H]TCDD photoaffinity labeled AHR. Specificity of the antiserum was confirmed by co-migration of photolabeled AHR with the major immunoreactive band identified by western blot. Further characterization showed that the antipeptide antibodies recognized equally both mouse and human AHR, which differ significantly in molecular mass (mouse Hepa-1 cells ≈ 95 kDa; human LS180 cells ≈ 110 kDa). The affinity-purified antibodies also recognized undenatured TCDD∙AHR complexes, as determined by a shift in sedimentation of the [3H]TCDD∙AHR complex on a sucrose gradient. The high specificity and sensitivity of this antibody were used to determine the fate of the AHR in cells exposed to [3H]TCDD. Western blot analysis revealed that TCDD exposure caused a dramatic decrease in total cellular AHR to about 20% pre-TCDD levels within 2 h after TCDD, which persisted up to 20 h after initial TCDD exposure. However, in the presence of actinomycin D or cycloheximide, nuclear AHR remained elevated in cells exposed to TCDD, at levels similar to or greater than the maximum previously observed after 1-h incubations. These data suggest that ligand-dependent downregulation of the AHR is the result of protein degradation by a short-lived protease.Key words: aromatic hydrocarbon receptor, polyclonal antibody, downregulation, 2,3,7,8-tetrachlorodibenzo

ISSN:0008-4212    

DOI:10.1139/y95-002

出版商:NRC Research Press    

年代:1995

数据来源: NRC

摘要:

In liver of adult responsive C57BL/6J (B6) mice the aromatic hydrocarbon receptor (AHR) has high affinity for specific halogenated aromatic hydrocarbons, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), as well as nonhalogenated aromatic hydrocarbons (PAHs), such as benz[a]anthracene (BA) or 3-methylcholanthrene (MC). In livers of adult nonresponsive DBA/2J (D2) mice TCDD binds to a low-affinity variant form of AHR. Both TCDD and MC induce aryl hydrocarbon hydroxylase (AHH) in adult B6 mice, whereas adult D2 mouse liver is nonresponsive to MC. In fetal cell cultures derived from D2 mice AHH is induced by PAHs such as MC or BA, and these PAHs bind to cytosolic AHR (P.A. Harper, C.L. Golas, and A.B. Okey. Mol. Pharmacol. 40: 818–826, 1991). We compared AHR from fetal cell cultures with AHR from adult livers to determine whether there was some structural difference in receptors expressed in fetal cell culture that might permit cells from "nonresponsive" mice to respond to PAHs. The apparent molecular mass of AHR from cells cultured from 18-day fetuses is identical with that from adult liver within each strain of inbred mice tested (Mr~ 95 kDa in B6 and ~ 105 kDa in D2 mice). The AHR in D2 fetal cells was able to activate a transfected chloramphenicol acetyltransferase linked to a dioxin-responsive element nucleotide sequence (DRE–CAT) when the cells were treated with TCDD or MC. The potency of CAT expression in D2 fetal cells was similar to that in B6 fetal cells. Our data suggest that the responsiveness of fetal cells from "nonresponsive" mice is likely mediated by AHR in these cells but is not due to expression of a different allelic form of AHR ligand-binding subunit in fetal cells versus adult liver.Key words: aromatic hydrocarbon receptor, 2,3,7,8-tetrachlorodibenzo-p-dioxin, cultured fetal cells, C57BL/6J mice, DBA/2J mice.

ISSN:0008-4212    

DOI:10.1139/y95-003

出版商:NRC Research Press    

年代:1995

数据来源: NRC

摘要:

The properties of Ca2+-mediated inactivation as revealed by a conventional double-pulse protocol were examined by using the whole-cell patch clamp technique. A U-shaped relationship between the conditioning potential and the Ca2+current (ICa) inactivation was observed, with a maximum inactivation of 52 ± 4% (n = 5) at 10 mV with 0.5 mM EGTA in the patch pipettes. The maximum inactivation was reduced significantly, to 31 ± 5.7% (n = 12) and 32 ± 7.0% (n = 5), when a high concentration of EGTA (20 mM) or a more efficient Ca2+chelator, BAPTA, was included in the patch pipettes, respectively. The same double-pulse protocol was applied under conditions where the stored Ca2+was depleted by using caffeine or the stored Ca2+release function was blocked by using ryanodine or procaine and heparin. No significant difference in the maximumICainactivation before (45%) and after (50%) application of 10 mM caffeine was observed. The maximumICainactivations of 48 ± 3.2% (n = 4) and 52 ± 8.4% (n = 6) were still observed after treatment of the cell with ryanodine (20 μM) or loading 10 mM procaine and 1 mg/mL heparin in the patch pipettes, respectively. These results suggest that Ca2+mobilization from an internal Ca2+store is not essential for the Ca2+-mediated inactivation observed in the double-pulse experiment, rather influx of Ca2+through a voltage-dependent Ca channel seems to be important forICainactivation. Recovery from Ca2+-mediated inactivation could be fitted by the sum of two exponentials; the time constants of the fast and slow components were 210 and 810 ms, respectively, at a holding potential of −80 mV. By lowering the holding potential to −30 from −80 mV, the time constants of both fast and slow components were increased to 740 and 4820 ms, respectively, suggesting that the recovery of Ca2+channels from Ca2+-mediated inactivation is voltage dependent.Key words: urinary bladder, smooth muscle, whole-cell patch clamp, Ca2+current, double-pulse protocol, Ca2+-mediated inactivation.

ISSN:0008-4212    

DOI:10.1139/y95-004

出版商:NRC Research Press    

年代:1995

数据来源: NRC

摘要:

We studied the effects of Zn2+and Cd2+on the behaviour ofIK(f), a transient outward potassium current in acutely dissociated melanotrophs of the pars intermedia of the rat pituitary gland. Micromolar concentrations of external Cd2+or Zn2+caused parallel and nearly equal rightward shifts along the voltage axis of the activation and steady-state inactivation curves forIK(f). TheKDfor the half-maximal shift of the activation curve was 278 μM for Cd2+and 93 μM for Zn2+; the maximal shifts of the activation curve were 32.5 and 34 mV, for Cd2+and Zn2+, respectively. The times to half-activation and half-inactivation were shifted rightward by 30–60 mV in both 500 μM Cd2+and 500 μM Zn2+. We suggest that Cd2+and Zn2+interact specifically with a binding site on or electrically close to theIK(f) channel and in so doing modify the electric field "seen" by the voltage sensors.Key words: fast transient K+current, melanotrophs, divalent cations, activation gating, inactivation gating.

ISSN:0008-4212    

DOI:10.1139/y95-005

出版商:NRC Research Press    

年代:1995

数据来源: NRC

摘要:

Effects of synthetic eel (E-), salmon (S-), and human (H-) calcitonin (CT) on gastrointestinal motility were studied in conscious beagle dogs, which had been implanted with strain gauge force transducers. Intramuscular administration of E-, S-, or H-CT interrupted gastric migrating motor complexes, digestive pattern, and gastric emptying. The order of potency was E-CT = S-CT > H-CT. Motor inhibition induced by CT occurred independently of plasma immunoreactive motilin levels or hypocalcemia. In addition, E-CT and S-CT induced vomiting without a retrograde giant contraction (RGC) during the postprandial state. Apomorphine or CuSO4initiated RGC prior to vomiting. RGC induced by apomorphine was inhibited by pretreatment with E-CT as well as hexamethonium, atropine, or surgical vagotomy. E-CT showed no inhibitory effect on nicotine stimulated contraction of isolated guinea-pig ileum. These results suggest that peripherally administered CT inhibits canine gastrointestinal motility at the central nervous system level by lowering vagal activity.Key words: gastric emptying, motilin, retrograde giant contraction, vagus, vomiting.

ISSN:0008-4212    

DOI:10.1139/y95-006

出版商:NRC Research Press    

年代:1995

数据来源: NRC

摘要:

The purpose of the present study was to estimate the development of the inotropic response to low extracellular sodium (LES) during the perinatal period. The effect of LES (35 mmol∙L−1) was measured in isolated perfused control and ryanodine-pretreated rat hearts on prenatal day 20 and postnatal days 1, 2, 4, and 7. The effect of LES on the developed force (DF) of control hearts changes significantly day by day: whereas a persisting increase of magnitude of contractions was recorded in the prenatal hearts, this increase was only transient on postnatal day 1 and 2. Starting from day 4, the initial signs of a triphasic response, typical for adult hearts, appeared (an initial increase of DF, followed by a decrease of DF and a rise of resting force, and finally a delayed increase of DF); this trend was more pronounced on day 7. The LES-induced increase of resting force was recorded only in 2-, 4-, and 7-day-old hearts. The negative inotropic effect of ryanodine (10−6 mol∙L−1) was observed already prenatally (60% of the controls) and continued during the whole period of investigation; in contrast, a ryanodine-induced increase of resting force was recorded only postnatally. However, pretreatment with ryanodine abolished the day-by-day changes in the response to LES: in all the hearts studied, the first phase (initial increase of DF) was followed by a severe depression of the magnitude of contractions, together with increased resting force. Our data show significant age-dependent differences in the cardiac contractile response to LES. This response changes rapidly during the perinatal development, and it attains the adult pattern by the end of the 1st postnatal week in rats.Key words: low extracellular sodium, ryanodine, inotropic effect, contractile function, perinatal ontogeny, rat heart, Na+–Ca2+excha

ISSN:0008-4212    

DOI:10.1139/y95-007

出版商:NRC Research Press    

年代:1995

数据来源: NRC

摘要:

Numerous studies, both in vitro and in vivo, have demonstrated the insulin-mimetic properties of vanadium. Chronic oral administration of inorganic and organic compounds of both vanadium(IV) and vanadium(V) reduced plasma glucose levels and restored plasma lipid levels in streptozotocin-diabetic rats. We investigated the acute effects of both vanadyl sulfate and bis(maltolato)oxovanadium(IV) (BMOV), an organic vanadium compound, on plasma glucose levels by several routes of administration. Previous studies have shown that chronic administration of vanadyl sulfate has resulted in a sustained euglycemia following withdrawal of the drug. This effect was not observed following the chronic administration of BMOV; therefore, we investigated the effect of increasing the concentration of BMOV on the production of a sustained euglycemic response. An acute plasma glucose lowering effect was obtained with both vanadyl sulfate and BMOV when administered as a single dose by either oral gavage or intraperitoneal injection. In those animals that responded to vanadium treatment, plasma glucose levels were within the normal range within 2 to 6 h when given by i.p. injection or within 4 to 8 h when given by oral gavage. BMOV-treated rats that responded to treatment maintained the euglycemic effect for extended periods, ranging from 1 to 14 weeks following administration. However, vanadyl sulfate treated rats reverted to hyperglycemia within 12 to 24 h, depending on the route of administration. Intravenous administration of BMOV was effective in lowering plasma glucose levels only when administered by continuous infusion. An oral dose – response curve showed that BMOV was 2 to 3 times as potent as vanadyl sulfate. This difference in potency was observed with both oral and intraperitoneal administration, which suggests that the increase in potency with BMOV cannot be totally attributed to increased gastrointestinal absorption. Organic chelation of vanadium may facilitate uptake into vanadium-sensitive tissues. Chronic oral administration of higher concentrations of BMOV did not result in a sustained reduction in plasma glucose following withdrawal of the drug. All diabetic rats eventually responded to increased concentrations of BMOV with a restoration of plasma glucose levels to normal values; however, reversion to the hyperglycemic state occurred within 2 days of withdrawal of treatment. Chronic oral administration of BMOV did not produce a sustained euglycemic effect following withdrawal, but acute administrations of the compound by either oral gavage or intraperitoneal injection did produce a long-term reduction in plasma glucose levels. Rats treated chronically with vanadyl sulfate remained euglycemic even after the drug was withdrawn. However, acute treatment produced only a transient euglycemia.Key words: streptozotocin diabetic, acute, bis(maltolato)oxovanadium(IV), vanadyl sulfate, dose response.

ISSN:0008-4212    

DOI:10.1139/y95-008

出版商:NRC Research Press    

年代:1995

数据来源: NRC

摘要:

Adult rat parasympathetic ciliary ganglion (CG) neurons were retrogradely labelled by intraocular injection of the carbocyanine fluorescent dye 1,1-dioleyl-3,3,3′,3′-tetramethylindocarbocyanine methanesulfonate (DiI). Whole-cell and nystatin perforated patch recording techniques were then used to examine the electrophysiological properties of labelled CG neurons growing in primary culture. The resting membrane potential of CG neurons in dissociated cell culture was −50 ± 8 mV, and isolated neurons fired overshooting action potentials in response to depolarizing current injection. Voltage-clamp recordings of membrane currents revealed a transient tetrodotoxin-sensitive Na+inward current and both sustained and transient outward K+currents. Sustained outward K+current was reduced (55–77%) by 5 mM tetraethylammonium and to a lesser extent (42–46%) by superfusion with nominally Ca2+free external solution. Transient outward current was blocked by 100 μm 4-aminopyridine and exhibited steady-state inactivation at potentials depolarized to −50 mV. These data demonstrate that identified adult mammalian CG neurons can be successfully maintained in culture. Cultured CG neurons retain electrical excitability, with voltage-sensitive Na+and K+currents giving rise to action potentials.Key words: fluorescent tracer, patch clamp, neuron, ciliary ganglion.

ISSN:0008-4212    

DOI:10.1139/y95-009

出版商:NRC Research Press    

年代:1995

数据来源: NRC

摘要:

The objective of this experiment was to determine whether bilateral renal denervation (RD) prevents the elevation of blood pressure and cardiac hypertrophy characteristically induced by chronic exposure to cold. Four groups (nine male rats each) were used. The kidneys of two groups were bilaterally denervated, while the remaining two groups were sham operated. Systolic blood pressures of the four groups, measured indirectly from the tail, did not differ significantly during the control period and following RD. At this time, 1 RD and 1 sham-operated group was exposed to cold (5 °C, 41°F). The remaining RD and sham-operated groups were kept at 25 °C. Blood pressure of the cold-exposed, sham-operated group increased significantly during the 1st week of cold exposure (125 ± 2 mmHg; 1 mmHg = 133.3 Pa), and rose to 139 ± 4 mmHg by the 5th week, whereas the blood pressure of the RD group exposed to cold remained at the control level (116 ± 2 mmHg). Both RD and sham-operated cold-exposed groups developed cardiac hypertrophy with significantly increased resting heart rates compared with controls kept at 25 °C. Plasma renin activities and renal norepinephrine content of kidneys of both RD groups at 7 weeks after RD were significantly less than those of sham-operated controls, confirming that renal nerves had been severed. Thus, RD prevented the elevation of blood pressure induced by chronic exposure to cold but had no significant effect on cardiac hypertrophy.Key words: cold exposure, rats, blood pressure elevation, hypertension, renal denervation, plasma renin activity, renal norepinephrine content, cardiac hypertrophy.

ISSN:0008-4212    

DOI:10.1139/y95-010

出版商:NRC Research Press    

年代:1995

数据来源: NRC