Changes in plasma arginine vasopressin concentration during lactation in rats.

BACKGROUND AND PURPOSE: The objectives of the study reported here were to determine whether a change in the plasma arginine vasopressin (AVP) concentration occurred in lactating, compared with non-lactating rats and to examine the involvement of suckling with plasma AVP concentration. METHODS: Experiments were performed on 86 female Wister Imamichi rats, 12 weeks old at parturition, with fast lactation. On day 13 of lactation, AVP concentration and plasma osmotic pressure were measured in lactating and non-lactating rats. RESULTS: Plasma AVP concentration was always higher in rats of the lactating groups than in non-lactating controls (1.06 +/- 0.28 pg/ml), and a conspicuous increase in AVP concentration was seen during the postsuckling period (1.70-0.61 pg/ml before vs. 2.56 +/- 1.31 pg/ml after suckling, P < 0.05). Plasma osmotic pressure in lactating rats with 12 pups (296.6 +/- 5.2 mOsmol/kg x H2O) was lower than that in rats of the removed control groups (306.7 +/- 5.7 mOsmol/kg x H2O). CONCLUSION: On the basis of these results, it appears that "low plasma osmotic pressure-high AVP status" develops in the lactating period, similar to pregnancy, through resetting of the regulatory mechanism of the AVP system. It was concluded that suckling stimulation could release AVP, which could dilute the blood with water resulting in the increase in circulating blood volume.

Preliminary studies on hepatic carnitine palmitoyltransferase in dairy cattle with or without fatty liver.

The hepatic mitochondrial carnitine palmitoyltransferase (CPT) activity was measured by fluorimetric assay in dairy cows with or without fatty liver. CPT activities in 13 lactating cattle and in 6 non-lactating cows were 304.4+/-86.6 micromol CoA/min per g protein and 169.3+/-84.8 micromol CoA/min per g protein, respectively. This difference was significant (p < 0.05). CPT activities in early lactation (0-110 days after calving), mid-lactation (111-220 days after calving) and late lactation (over 220 days after calving) were 278.9+/-68.0, 312.4+/-124.1 and 320+/-59.3 micromol CoA/min per g protein, respectively. There was no significant difference between the values at different stages of lactation. The CPT activity in 10 lactating cows with fatty liver unrelated to calving was 201.3+/-80.0 micromol CoA/min per g protein. CPT activity in 10 cattle with fatty liver was significantly lower than that in normal lactating cattle. Based on these findings, clinical fatty liver unrelated to calving appears to be associated with a decrease in hepatic CPT activity.

Cellular characterization of pituitary adenoma cell line (AtT20 cell) transfected with insulin, glucose transporter type 2 (GLUT2) and glucokinase genes: insulin secretion in response to physiological concentrations of glucose.

We investigated the mechanisms of insulin secretion by transfecting into a pituitary adenoma cell line (AtT20) a combination of genes encoding human insulin (HI), glucose transporter type 2 (GLUT2) and glucokinase (GK), followed by studying the characteristics of these cells. In static incubation, a cell line transfected with insulin gene alone (AtT20HI) secreted mature human insulin but this was not in a glucose-dependent manner. Other cell lines transfected with insulin and GLUT2 genes (AtT20HI-GLUT2-3) or with insulin and GK genes (AtT20HI-GK-1) secreted insulin in response to glucose concentrations of only less than 1 mmol/l. In contrast, cell lines transfected with insulin, GLUT2 and GK genes (AtT20HI-GLUT2-GK-6, AtT20HI-GLUT2-GK-7 and AtT20HI-GLUT2-GK-10) showed a glucose-dependent insulin secretion up to 25 mmol/l glucose. Glucose utilization and oxidation were increased in AtT20HI-GLUT2-GK cell lines but not in AtT20HI, AtT20HI-GLUT2-3 and AtT20HI-GK-1 cells at physiological glucose concentrations, compared with AtT20 cells. Diazoxide, nifedipine and 2-deoxy glucose suppressed (p < 0.05) glucose stimulated insulin secretion in AtT20HI-GLUT2-GK-6 cells. Glibenclamide, KCl or corticotropin releasing factor (CRF) stimulated (p < 0.05) insulin secretion both in AtT20HI and AtT20HI-GLUT2-GK-6 cells. Insulin secretion stimulated by glibenclamide, KCl or CRF was further enhanced by the addition of 25 mmol/l glucose in AtT20HI-GLUT2-GK-6 cells but not in AtT20HI cells. In perifusion experiments, a stepwise increase in glucose concentration from 5 to 25 mmol/l stimulated insulin secretion in AtT20HI-GLUT2-GK cell lines but the response lacked a clear first phase of insulin secretion. Our results suggest that both GLUT2 and glucokinase are necessary for the glucose stimulated insulin secretion in at least rodent cell lines, and that other element(s) are necessary for a biphasic insulin secretion typically observed in beta cells.

Seroprevalence against Toxoplasma gondii in domiciled cats in Japan.

A serological survey with latex agglutination test to detect anti-Toxoplasma gondii antibodies was conducted on 800 serum samples collected from domiciled cats at animal hospitals in various areas of Japan. The overall prevalence was 6.0% (48/800). Among 48 positive individuals, there was no specific distribution of strength of antibody titers; the titers were 1:64 in 8 cats, 1:128 in 12, 1:256 in 8, 1:512 in 10, 1:1,024 in 8 and 1:2,048 in 2. The maximum prevalence was 15.4% in 13 cats at 17-23 yrs old group, whereas all were negative in 58 cats aged 12-16 yrs. The age groups in the order of higher prevalence were 8, 4, 10, 5, 3, and 7 yrs, showing no aging effect to the prevalence. In terms of rearing conditions of those cats, they were classified into 4 groups, i.e., indoor, free, outdoor, and others. The prevalence in the outdoor group (11.1%) was significantly higher (p < 0.05) than that in the free group (4.8%). Epidemiological aspects observed in the domiciled cats were different from those reported in the stray cats.

Model of emotional stress in rats.

A new experimental rat model for suppression of release of arginine vasopressin (AVP) was evaluated. Release of AVP is potentiated by physiologic stimuli, but is suppressed by emotional stress. Rats were exposed to the aggressive behavior of other rats injected with 6-hydroxydopamine hydrochloride (6-OHDA) in both lateral ventricles and subjected to a pain stimulus applied to the tail. To ascertain whether emotional stress is induced by use of this method, plasma corticosterone, glucose, and AVP concentrations were measured in the stressed group of rats (n = 8) placed near a group of aggressive (fighting) rats. Characteristic changes in behavior, significant increases in plasma corticosterone and glucose values, and lack of increase in plasma AVP concentration in stressed rats confirmed that they experienced emotional stress. This new model meets the experimental requirements for suppression of AVP release in rats.

Hepatic carnitine palmitoyltransferase activity in cattle.

A fluorometric assay for the determination of hepatic carnitine palmitoyltransferase (CPT) activity was slightly modified for use with cattle samples. With this assay, the Km value was 0.56 +/- 0.10 mM with respect to L-carnitine (mean +/- SD, n = 4) and was 9.6 +/- 2.2 microM (n = 3) with respect to palmitoyl-CoA. The average hepatic CPT activity was 33.6 +/- 2.0 mumol CoASH/min/g protein in 38 healthy cattle and was similar in both sexes and among breeds. Hepatic CPT activity showed no correlation with serum phospholipid, free fatty acid, triglyceride or total cholesterol concentrations.

Serum concentration of circulating immune complexes in cats infected with feline immunodeficiency virus detected by immune adherence hemagglutination method.

Circulating immune complexes (CIC) in the sera from 45 clinically healthy cats and 23 feline immunodeficiency virus (FIV) infected cats were measured by an immune adherence hemagglutination (IAHA) method. The level of CIC in the sera from FIV sera-negative healthy cats was 47.2 +/- 47.3 micrograms/ml when expressed as heat aggregated feline IgG equivalent value in IAHA reactivity. On the other hand, the level of CIC in the sera from FIV infected cats was 757.4 +/- 910.5 micrograms/ml, which was significantly higher than that of healthy ones. CIC levels of 11 symptomatic cats and 12 asymptomatic ones were 837.8 +/- 1138.2 micrograms/ml and 683.0 +/- 684.2 micrograms/ml, respectively. These results showed that IAHA method was reliable to detect CIC levels of cat sera and that CIC levels in the sera of cats infected with FIV were higher than those of healthy ones.

Blood sampling technique for measurement of plasma arginine vasopressin concentration in conscious and unrestrained rats.

A new technique for collecting blood from conscious and unrestrained rats is reported. Plasma arginine vasopressin (AVP) concentration has been known to be influenced by various procedures used for blood collection (e.g., manual contact and anesthesia). In the study reported here, the improvement of the routine jugular vein cannula method was undertaken by extending the connection tubing to allow use of a swivel mechanism. Plasma AVP, glucose, and corticosterone concentrations in virgin rats were measured in blood samples collected by use of the new method and of two generally accepted methods (decapitation and routine jugular vein cannula methods). All values of the three parameters obtained were the lowest when the new method was used, suggesting that this new swivel method induced the least stress in the rats.

Decreased pulmonary arterial endothelium-dependent relaxation in heartworm-infected dogs with pulmonary hypertension.

OBJECTIVE: To investigate the contractility of pulmonary arterial smooth muscle and the relation between pulmonary hypertension and endothelium-derived relaxing factor in canine heartworm disease. ANIMALS: 18 noninfected control and 9 heartworm-infected dogs. PROCEDURE: Mean pulmonary arterial blood pressure was measured in vivo, and tension of pulmonary arterial strips was measured by use of the isometric tension method. RESULTS: After phenylephrine (10(-5)M)-induced contraction of the pulmonary vascular smooth muscle, carbamylcholine chloride (CCh, 10(-6)M) caused more relaxation of the vascular smooth muscle of noninfected, dogs than that of heartworm-infected dogs. Furthermore, the degree of CCh-induced relaxation was inversely correlated with mean pulmonary arterial blood pressure in the noninfected and the heartworm-infected dogs. The CCh-induced relaxation was inhibited by pretreatment with NG-nitro-L-arginine methyl ester hydrochloride (10(-5)M), and in reversed dose-dependent manner by L-arginine (10(-4) to 3 x 10(-2)M). Sodium nitroprusside (10(-8) to 10(-5)M) caused a dose-dependent relaxation in all vessels, and there was no significant difference in the relaxation responses in both groups except at 10(-7)M for vessels with intact endothelium from noninfected dogs. CONCLUSION: The depression of endothelium-dependent relaxation is correlated with the pulmonary arterial blood pressure in heartworm-infected dogs, suggesting that the decrease is one of the essential factors for the genesis of pulmonary hypertension in canine filariasis.

Detection of circulating immune complexes in dog sera by immune adherence hemagglutination method.

Circulating immune complexes (CIC) in the sera from 102 clinically healthy dogs and from 16 Dirofilaria immitis (D. immitis) infected dogs were measured by the immune adherence hemagglutination (IAHA) method. The level of CIC in the sera from healthy dogs was 28.2 +/- 29.1 micrograms/ml. There was no significant difference in the levels of CIC regarding sexes or ages. On the other hand, the level of CIC in the sera from D. immitis infected dogs was 230.3 +/- 117.1 micrograms/ml, which was significantly (p < 0.01) higher than that of healthy dogs. The results of this study confirm that IAHA method is one of the reliable means of detecting and/or diagnosing immune complex mediated diseases in dogs.

Multifocal ventricular tachycardia after resuscitation by direct current counter shock in a dog.

Cardiac arrest occurred in a male Labrador Retriever dog weighing 27.8 kg during induction to anesthesia. Immediately after the failure of resuscitation by the external cardiac compression, thoracotomy was performed and open chest direct current (DC) counter shocks were applied with routine emergency medications. Then the dog recovered consciousness. Although cardiac rhythm just after resuscitation was sinus tachycardia with paroxysmal supraventricular tachycardia, multifocal ventricular arrhythmia occurred 2 hr after resuscitation. This arrhythmia might be the result from reversible cardiac lesions due to DC counter shock.

Molecular scanning of the insulin receptor substrate-1 (IRS-1) gene in Japanese patients with NIDDM: identification of five novel polymorphisms.

Since the insulin receptor substrate-1 (IRS-1) is the major substrate of the insulin receptor tyrosine kinase and has been shown to activate phosphatidylinositol (PI) 3-kinase and promote GLUT4 translocation, the IRS-1 gene is a potential candidate for development of non-insulin-dependent diabetes mellitus (NIDDM). In this study, we have identified IRS-1 gene polymorphisms, evaluated their frequencies in Japanese subjects, and analysed the contribution of these polymorphisms to the development of NIDDM. The entire coding region of the IRS-1 gene of 94 subjects (47 NIDDM and 47 control subjects) was screened by polymerase chain reaction-single stranded conformation polymorphism (PCR-SSCP) analysis. Seven SSCP polymorphisms were identified. These corresponded to two previously identified polymorphisms [Gly971 --> Arg (GGG --> AGG) and Ala804 (GCA --> GCG)] as well as five novel polymorphisms [Pro190 --> Arg (CCC --> CGC), Met209 --> Thr (ATG --> ACG), Ser809 --> Phe (TCT --> TTT), Leu142 (CTT --> CTC), and Gly625 (GGC --> GGT)]. Although the prevalence of each of these polymorphisms was not statistically different between NIDDM and control subjects, the prevalence of the four IRS-1 polymorphisms with an amino acid substitution together was significantly higher in NIDDM than in control subjects (23.4 vs 8.5%, p < 0.05), and two substitutions (Met 209 --> Thr and Ser809 --> Phe) were found only in NIDDM patients. Equilibrium glucose infusion rates during a euglycaemic clamp in NIDDM and control subjects with the IRS-1 polymorphisms decreased by 29.5 and 22.0%, respectively on the average when compared to those in comparable groups without polymorphisms, although they were not statistically significant. Thus, IRS-1 polymorphisms may contribute in part to the insulin resistance and development of NIDDM in Japanese subjects; however, they do not account for the major part of the decrease in insulin-stimulated glucose uptake which is observed in subjects with clinically apparent NIDDM.

Bradykinin enhances GLUT4 translocation through the increase of insulin receptor tyrosine kinase in primary adipocytes: evidence that bradykinin stimulates the insulin signalling pathway.

It has been suggested that bradykinin stimulates glucose uptake in experiments in vivo and in cultured cells. However, its mechanism has not yet been fully elucidated. In this study, the effects of bradykinin on the insulin signalling pathway were evaluated in isolated dog adipocytes. The bradykinin receptor binding study revealed that dog adipocytes possessed significant numbers of bradykinin receptors (Kd = 83 pmol/l, binding sites = 1.7 x 10(4) site/ cell). Reverse transcription-polymerase chain reaction amplification showed the mRNA specific for bradykinin B2 receptor in the adipocytes. Bradykinin alone did not increase 2-deoxyglucose uptake in adipocytes; however, in the presence of insulin (10(-7) mol/l) it significantly increased 2-deoxyglucose uptake in a dose-dependent manner. Bradykinin also enhanced insulin stimulated GLUT4 translocation from the intracellular fraction to the cell membrane, and insulin induced phosphorylation of the insulin receptor beta subunit and insulin receptor substrate-1 (IRS-1) without affecting the binding affinities or numbers of cell surface insulin receptors in dog adipocytes. The time-course of insulin stimulated phosphorylation of the insulin receptor beta subunit revealed that phosphorylation reached significantly higher levels at 10 min, and stayed at the higher levels until 120 min in the presence of bradykinin, suggesting that bradykinin delayed the dephosphorylation of the insulin receptor. It is concluded that bradykinin could potentiate insulin induced glucose uptake through GLUT4 translocation. This effect could be explained by the potency of bradykinin to upregulate the insulin receptor tyrosine kinase activity which stimulates phosphorylation of IRS-1, followed by GLUT4 translocation.

Congestive heart failure due to bilateral atrioventricular valve insufficiency in two dogs.

Congestive heart failure resulted from bilateral atrioventricular insufficiency was diagnosed in two, small breed, intact male aged dogs. In both cases, the clinical signs were mainly associated with right heart failure, while those related to left heart failure were seldom observed. Radiographic and echocardiographic examinations revealed the dilatation of the right heart due to tricuspid valve insufficiency. Mitral regurgitation was also observed without pulmonary hypertension. Both patients responded to medications of diuretics, methyldigoxin and angiotensin converting enzyme inhibitor. As congestive signs deteriorated, they became unresponsive to the treatment. Ultimately, the dogs died 9 and 13 months later from the first admission.

Longitudinal distribution of pulmonary vascular compliance in dogs.

The longitudinal distribution of pulmonary vascular compliance was evaluated in isolated canine lung lobes using arterial-(AO), venous-(VO), and double-occlusion (DO) techniques. Total vascular compliance (Ctau) was separated into pulmonary arterial (Ca) and venous compliance (Cv) in lumped model of pulmonary circulation. Under constant pulmonary venous pressure (Pv) at 5 mmHg, blood inflow to the lobe (Q) was gradually increased by changing pulmonary arterial pressure (Pa) from 10 to 22 mmHg at 4 mmHg ranges. Changes in vascular blood volume (deltaV) with each increment in Q were determined by decreased reservoir blood volume of perfusion system. DO was performed at each level of Q and allowing all vascular pressures to equilibrate at the same static pressure (Ps), which was equal to the compliance-weighted average pressure in the circulation. Ctau was obtained from the slope of the relationship between Ps and deltaV. When Pa and Pv were 14 and 5 mmHg, AO, VO, and DO were performed to measure pressures at Ca (Pca) and Cv (Pcv) and Ps. The arterial-to-venous compliance ratio (Ca/Cv) was evaluated using Pca, Pcv, and Ps measurements. Ctau was 0.113 +/- 0.012 ml/kg/mmHg. Ca/Cv was 0.30. Ca and Cv were 0.026 +/- 0.013 and 0.087 +/- 0.007 ml/kg/mmHg, respectively. These data demonstrated the usefulness of AO, VO, and DO techniques in evaluating the longitudinal distribution of compliance in canine pulmonary vasculature.

Use of enzyme-linked-immunosorbent assay for detection of IgG and IgM antibodies to Fusobacterium necrophorum in cattle.

An enzyme-linked-immunosorbent assay (ELISA) with HCl heat-extracted antigen of Fusobacterium necrophorum was conducted to detect specific immunoglobulins G and M in infected cattle. The ELISA revealed an increase (> 0.40) in specific IgG in most of the animals with hepatic abscesses but not that in specific IgM. All the lesions were positive for F. necrophorum. These findings indicated that the ELISA for immunoglobulin G detection may prove to be a useful tool for predictive serodiagnosis of F. necrophorum infection in cattle.

The effect of hypovolemic shock and reperfusion on the hepatic oxygen supply-uptake relationship in the dog.

The hepatic oxygen supply-uptake relationship was investigated during hypovolemic shock using a right heart bypass technique. The results were dissimilar to those previously reported in that the ratio of liver oxygen delivery to systemic oxygen delivery was significantly decreased during shock. The decreased ratio was due to a significant decrease in the portal venous oxygen delivery when compared to the decrease in the systemic oxygen delivery. The decrease in portal venous oxygen delivery was caused not only by the decrease in portal venous blood flow, but also by the decrease in oxygen content of portal blood. The ratio of hepatic arterial oxygen delivery, on the other hand, was significantly increased during shock. Hypovolemic shock increased the liver oxygen extraction ratio to nearly 100% of the pre-shock value. These findings suggest a hepatic protective mechanism for matching oxygen uptake to rising hepatic oxygen requirements. Liver oxygen delivery returned to pre-shock value after correction of hypovolemia primarily due to a significant increase in hepatic arterial oxygen delivery. A significant negative correlation between the liver oxygen extraction ratio and the oxygen content of hepatic venous blood was observed. The hepatic venous oxygen content appears to be a simple and appropriate index of liver oxygenation in clinical medicine because it is difficult to evaluate the liver oxygen extraction ratio directly.

Effects of hypovolemic shock and reperfusion on liver blood flow in the dog.

Liver blood flow was investigated in hypovolemic shock using a modified right heart bypass technique which can obtain accurate portal blood flow. Findings were similar to those previously reported: hepatic blood flow accounted for 34% of cardiac output in this study; 76% of hepatic blood flow was delivered from the portal vein and 24% from the hepatic artery. Hypovolemic shock markedly decreased total liver blood flow by a reduction in portal venous blood flow. The findings of this study provide evidence that mesenteric blood flow is a peripheral circulation circuit where blood flow is restricted during reduced circulatory volume. Development of a hepatic arterial buffer response during hypovolemic shock was confirmed by an increased ratio of hepatic arterial flow to cardiac output. Reduced total hepatic blood flow during hypovolemic shock returned to control flow by an increase in hepatic arterial flow after reperfusion. The results of this study demonstrate that compensated reactions for maintaining liver blood flow mainly due to the hepatic arterial buffer response were functioned both during hypovolemic shock and after elimination of shock.

Intensive insulin therapy prevents the progression of diabetic microvascular complications in Japanese patients with non-insulin-dependent diabetes mellitus: a randomized prospective 6-year study.

To examine whether intensive glycemic control could decrease the frequency or severity of diabetic microvascular complications, we performed a prospective study of Japanese patients with non-insulin-dependent diabetes mellitus (NIDDM) treated with multiple insulin injection treatment. A total of 110 patients with NIDDM was randomly assigned to multiple insulin injection treatment group (MIT group) or to conventional insulin injection treatment group (CIT group). Fifty-five NIDDM patients who showed no retinopathy and urinary albumin excretions < 30 mg/24 h at the baseline were evaluated in the primary-prevention cohort, and the other 55 NIDDM patients who showed simple retinopathy and urinary albumin excretions < 300 mg/24 h were evaluated in the secondary-intervention cohort. The appearance and the progression of retinopathy, nephropathy and neuropathy were evaluated every 6 months over a 6-year period. The worsening of complications in this study was defined as an increase of 2 or more steps in the 19 stages of the modified ETDRS interim scale for retinopathy and an increase of one or more steps in 3 stages (normoalbuminuria, microalbuminuria and albuminuria) for nephropathy. The cumulative percentages of the development and the progression in retinopathy after 6 years were 7.7% for the MIT group and 32.0% for the CIT group in the primary-prevention cohort (P = 0.039), and 19.2% for MIT group and 44.0% for CIT group in the secondary-intervention cohort (P = 0.049). The cumulative percentages of the development and the progression in nephropathy after 6 years were 7.7% for the MIT group and 28.0% for the CIT group in the primary-prevention cohort (P = 0.032), and 11.5% and 32.0%, respectively, for the MIT and CIT groups in the secondary-intervention cohort (P = 0.044). In neurological tests after 6 years, MIT group showed significant improvement in the nerve conduction velocities, while the CIT group showed significant deterioration in the median nerve conduction velocities and vibration threshold. Although both postural hypotension and the coefficient of variation of R-R interval tended to improve in the MIT group, they deteriorated in the CIT group. In conclusion, intensive glycemic control by multiple insulin injection therapy can delay the onset and the progression of diabetic retinopathy, nephropathy and neuropathy in Japanese patients with NIDDM. From this study, the glycemic threshold to prevent the onset and the progression of diabetic microangiopathy is indicated as follows; HbA1c < 6.5%, FBG < 110 mg/dl, and 2-h post-prandial blood glucose concentration < 180 mg/dl.

DNase I interaction on muscle Z-line.

The effect of deoxyribonuclease I on muscle Z-line structures was re-examined. Under conditions of deoxyribonuclease I activation (presence of the divalent cation Ca2+ and Mg2+), a deoxyribonuclease I preparation did not affect Z-line structure if phenylmethylsulfonylfluoride, an inhibitor of serine proteases, was also present. In the absence of protease inhibitor, both Z-lines and M-lines were digested, even in the presence of EDTA and EGTA as inhibitors of deoxyribonuclease I. These electron microscopic observations were consistent with the following results from sodium dodecyl sulphate gel electrophoresis: when the protease was inhibited but deoxyribonuclease I was activated, myofibrillar proteins remained essentially intact. However, degradation of proteins in both rabbit psoas and chicken pectoralis myofibrils was observed in the presence of deoxyribonuclease I inhibitors when the protease inhibitor was absent. Our data strongly suggest that the interaction of deoxyribonuclease I with Z-line proteins previously reported is most likely due to contamination of the deoxyribonuclease I fraction by the serine-type proteases.