Intra-organ cell specific mitochondrial quantitative interactomics.

Almost every organ consists of many cell types, each with its unique functions. Proteomes of these cell types are thus unique too. But it is reasonable to assume that interactome (inter and intra molecular interactions of proteins) are also distinct since protein interactions are what ultimately carry out the function. Podocytes and tubules are two cell types within kidney with vastly different functions: podocytes envelop the blood vessels in the glomerulus and act as filters while tubules are located downstream of the glomerulus and are responsible for reabsorption of important nutrients. It has been long known that for tubules mitochondria plays an important role as they require a lot of energy to carry out their functions. In podocytes, however, it has been assumed that mitochondria might not matter as much in both normal physiology and pathology1. Here we have applied quantitative cross-linking mass spectrometry to compare mitochondrial interactomes of tubules and podocytes using a transgenic mitochondrial tagging strategy to immunoprecipitate cell-specific mitochondria directly from whole kidney. We have uncovered that mitochondrial proteomes of these cell types are quite similar, although still showing unique features that correspond to known functions, such as high energy production through TCA cycle in tubules and requirements for detoxification in podocytes which are on the frontline of filtration where they encounter toxic compounds and therefore, as a non-renewing cell type they must have ways to protect themselves from cellular toxicity. But we gained much deeper insight with the interactomics data. We were able to find pathways differentially regulated in podocytes and tubules based on changing cross-link levels and not just protein levels. Among these pathways are betaine metabolism, lysine degradation, and many others. We have also demonstrated how quantitative interactomics could be used to detect different activity levels of an enzyme even when protein abundances of it are the same between cell types. We have validated this finding with an orthogonal activity assay. Overall, this work presents a new view of mitochondrial biology for two important, but functionally distinct, cell types within the mouse kidney showing both similarities and unique features. This data can continue to be explored to find new aspects of mitochondrial biology, especially in podocytes, where mitochondria has been understudied. In the future this methodology can also be applied to other organs to uncover differences in the function of cell types.

Estimated glucose disposal rate as a candidate biomarker for thrombotic biomarkers in T1D: a pooled analysis.

PURPOSE: To determine the utility of estimated glucose disposal rate (eGDR) as a candidate biomarker for thrombotic biomarkers in patients with type 1 diabetes (T1D). METHODS: We reanalysed baseline pretreatment data in a subset of patients with T1D from two previous RCTs, consisting of a panel of thrombotic markers, including fibrinogen, tissue factor (TF) activity, and plasminogen-activator inhibitor (PAI)-1, and TNFα, and clinical factors (age, T1D duration, HbA1c, insulin requirements, BMI, blood pressure, and eGDR). We employed univariate linear regression models to investigate associations between clinical parameters and eGDR with thrombotic biomarkers. RESULTS: Thirty-two patients were included [mean ± SD age 31 ± 7 years, HbA1c of 58 ± 9 mmol/mol (7.5 ± 0.8%), eGDR 7.73 ± 2.61]. eGDR negatively associated with fibrinogen (P < 0.001), PAI-1 concentrations (P = 0.005), and TF activity (P = 0.020), but not TNFα levels (P = 0.881). We identified 2 clusters of patients displaying significantly different characteristics; 56% (n = 18) were categorised as 'higher-risk', eliciting significantly higher fibrinogen (+ 1514 ± 594 µg/mL; P < 0.001), TF activity (+ 59.23 ± 9.42 pmol/mL; P < 0.001), and PAI-1 (+ 8.48 ± 1.58 pmol/dL; P < 0.001), HbA1c concentrations (+ 14.20 ± 1.04 mmol/mol; P < 0.001), age (+ 7 ± 3 years; P < 0.001), duration of diabetes (15 ± 2 years; P < 0.001), BMI (+ 7.66 ± 2.61 kg/m2; P < 0.001), and lower mean eGDR (- 3.98 ± 1.07; P < 0.001). CONCLUSIONS: Compared to BMI and insulin requirements, classical surrogates of insulin resistance, eGDR is a suitable and superior thrombotic risk indicator in T1D. TRIAL REGISTRATION: ISRCTN4081115; registered 27 June 2017.

Hip resurfacing as an outpatient procedure: a comparison of overall cost and review of safety.

Recent advancements in arthroplasty surgical techniques and perioperative protocols have reduced the duration of hospitalization and length of recovery, allowing surgeons to perform joint replacement as an outpatient procedure. This study aims to evaluate the cost-effectiveness and safety of outpatient hip resurfacing. Two experienced surgeons performed 485 resurfacing surgeries. We retrospectively compared clinical outcomes and patient satisfaction with published outpatient total hip results. Furthermore, we compared average insurance reimbursement with that of local inpatient hip replacement. No major complications occurred within 6 weeks. Of the 39 patients with previous inpatient experience, 37 (95%) believed their outpatient experience was superior. The average reimbursement for hip arthroplasty at local hospitals was $50,000, while the average payment for outpatient resurfacing at our surgery center was $26,000. We conclude that outpatient hip resurfacing can be accomplished safely, with high patient satisfaction, and at a tremendous financial savings to the insurer/patient. LEVEL OF EVIDENCE: III.

Gut microbiota of Type 1 diabetes patients with good glycaemic control and high physical fitness is similar to people without diabetes: an observational study.

AIM: Type 1 diabetes is the product of a complex interplay between genetic susceptibility and exposure to environmental factors. Existing bacterial profiling studies focus on people who are most at risk at the time of diagnosis; there are limited data on the gut microbiota of people with long-standing Type 1 diabetes. This study compared the gut microbiota of patients with Type 1 diabetes and good glycaemic control and high levels of physical-fitness with that of matched controls without diabetes. METHODS: Ten males with Type 1 diabetes and ten matched controls without diabetes were recruited; groups were matched for gender, age, BMI, peak oxygen uptake (VO2max ), and exercise habits. Stool samples were analysed using next-generation sequencing of the 16S rRNA gene to obtain bacterial profiles from each individual. Phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) was implemented to predict the functional content of the bacterial operational taxonomic units. RESULTS: Faecalibacterium sp., Roseburia sp. and Bacteroides sp. were typically the most abundant members of the community in both patients with Type 1 diabetes and controls, and were present in every sample in the cohort. Each bacterial profile was relatively individual and no significant difference was reported between the bacterial profiles or the Shannon diversity indices of Type 1 diabetes compared with controls. The functional profiles were more conserved and the Type 1 diabetes group were comparable with the control group. CONCLUSIONS: We show that both gut microbiota and resulting functional bacterial profiles from patients with long-standing Type 1 diabetes in good glycaemic control and high physical fitness levels are comparable with those of matched people without diabetes.

Algorithm that delivers an individualized rapid-acting insulin dose after morning resistance exercise counters post-exercise hyperglycaemia in people with Type 1 diabetes.

AIMS: To develop an algorithm that delivers an individualized dose of rapid-acting insulin after morning resistance exercise to counter post-exercise hyperglycaemia in individuals with Type 1 diabetes. METHODS: Eight people with Type 1 diabetes, aged 34 ± 7 years with HbA1c concentrations 72 ± 12 mmol/mol (8.7 ± 1.1%), attended our laboratory on two separate mornings after fasting, having taken their usual basal insulin the previous evening. These people performed a resistance exercise session comprising six exercises for two sets of 10 repetitions at 60% of the maximum amount of force that was generated in one maximal contraction (60% 1RM). In a randomized and counterbalanced order, the participants were administered an individualized dose of rapid-acting insulin (2 ± 1 units, range 0-4 units) immediately after resistance exercise (insulin session) by means of an algorithm or were not administered this (no-insulin session). Venous blood glucose concentrations were measured for 125 min after resistance exercise. Data (mean ± sem values) were analysed using anova (P ≤ 0.05). RESULTS: Participants had immediate post-resistance exercise hyperglycaemia (insulin session 13.0 ± 1.6 vs. no-insulin session 12.7 ± 1.5 mmol/l; P = 0.834). The decline in blood glucose concentration between peak and 125 min after exercise was greater in the insulin exercise session than in the no-insulin session (3.3 ± 1.0 vs. 1.3 ± 0.4 mmol/l: P = 0.015). There were no episodes of hypoglycaemia (blood glucose <3.9 mmol/l). CONCLUSIONS: Administration of rapid-acting insulin according to an individualized algorithm reduced the hyperglycaemia associated with morning resistance exercise without causing hypoglycaemia in the 2 h post-exercise period in people with Type 1 diabetes.

Similar magnitude of post-exercise hyperglycemia despite manipulating resistance exercise intensity in type 1 diabetes individuals.

The aim of this study was to compare the glycemic and glucoregulatory hormone responses to low- and moderate-intensity morning resistance exercise (RE) sessions in type 1 diabetes (T1DM). Following maximal strength assessments (1RM), eight T1DM (HbA1C :72 ± 12 mmol/mol, age:34 ± 7 years, body mass index:25.7 ± 1.6 kg/m(2) ) participants attended the research facility on two separate occasions, having fasted and taken their usual basal insulin but omitting rapid-acting insulin. Participants performed six exercises for two sets of 20 repetitions at 30%1RM during one session [low-intensity RE session (LOW)] and two sets of 10 repetitions at 60%1RM during another session [moderate-intensity RE session (MOD)], followed by 65-min recovery. Sessions were matched for total mass lifted (kg). Venous blood samples were taken before and after exercise. Data (mean ± SEM) were analyzed using analysis of variance (P ≤ 0.05). There were no hypoglycemic occurrences throughout the study. Blood glucose rose similarly between sessions during exercise (P = 0.382), remaining comparable between sessions throughout recovery (P > 0.05). There was no effect of RE intensity on metabolic acidosis (P > 0.05) or peak growth hormone responses (P = 0.644), but a tendency for greater catecholamine responses under LOW (individualized peak concentrations: adrenaline MOD 0.55 ± 0.13 vs LOW 1.04 ± 0.37 nmol/L, P = 0.155; noradrenaline MOD 4.59 ± 0.86 vs LOW 7.11 ± 1.82 nmol/L, P = 0.082). The magnitude of post-exercise hyperglycemia does not differ between equal volume low and moderate intensity RE sessions performed in the morning.

Simulated games activity vs continuous running exercise: a novel comparison of the glycemic and metabolic responses in T1DM patients.

To compare the glycemic and metabolic responses to simulated intermittent games activity and continuous running exercise in type 1 diabetes. Nine patients (seven male, two female; 35 ± 4 years; HbA1c 8.1 ± 0.2%/65 ± 2 mmol/mol) treated on a basal-bolus regimen completed two main trials, a continuous treadmill run (CON) or an intermittent running protocol (INT). Patients arrived to the laboratory fasted at ∼ 08:00 h, replicating their usual pre-exercise meal and administering a 50% reduced dose of rapid-acting insulin before exercising. Blood glucose (BG), K(+) , Na(++) , pH, triglycerides, serum cortisol and NEFA were measured at baseline and for 60 min post-exercise. Interstitial glucose was measured for a further 23 h under free-living conditions. Following exercise, BG declined under both conditions but was less under INT (INT -1.1 ± 1.4 vs CON -5.3 ± 0.4 mmol/L, P = 0.037), meaning more patients experienced hypoglycemia (BG ≤ 3.5 mmol/L; CON n = 3 vs INT n = 2) but less hyperglycemia (BG ≥ 10.9 mmol/L; CON n = 0 vs INT n = 6) under CON. Blood lactate was significantly greater, and pH lower, with a temporal delay in K(+) under INT (P < 0.05). No conditional differences were observed in other measures during this time, or in interstitial glucose concentrations during the remaining 23 h after exercise. Simulated games activity carries a lower risk of early, but not late-onset hypoglycemia than continuous running exercise in type 1 diabetes.

Impact of single and multiple sets of resistance exercise in type 1 diabetes.

To examine glycemic and glucoregulatory responses to resistance exercise (RE) sessions of different volume in type 1 diabetes (T1DM). Eight T1DM (seven males: one female; age: 38 ± 6 years, HbA1C : 8.7 ± 1.0%/71 ± 11 mmol/mol) attended the research facility fasted and on four separate occasions, having taken their usual basal insulin, but omitted morning rapid-acting insulin. Participants completed a 1SET (14 min), 2SET (28 min), 3SET (42 min) RE session (eight exercises × 10 repetitions) at 67 ± 3% one-repetition-maximum followed by 60-min recovery, or a resting trial (CON). Venous blood samples were taken before and after exercise. Data (mean ± SEM) were analyzed using repeated-measures analysis of variance (P ≤ 0.05). RE did not induce hypoglycemia (BG < 4 mmol/L). During recovery, blood glucose (BG) concentrations remained above pre-exercise after 1SET (15-60 min, P < 0.05) and 2SET (0-60 min, P < 0.05) but comparable (P > 0.05) with pre-exercise after 3SET. BGIAUC(area-under-curve) (mmol/L/60 min) was greater after 1SET and 2SET vs CON (1SET 103.6 ± 36.9 and 2SET 128.7 ± 26.1 vs CON -24.3 ± 15.2, P < 0.05), but similar between 3SET and CON (3SET 40.7 ± 59.3, P > 0.05). Under all trials, plasma creatine kinase levels at 24 h post-exercise were similar (P > 0.05) to pre-exercise. RE does not induce acute hypoglycemia or damage muscle. BG progressively rose after one and two sets of RE. However, inclusion of a third set attenuated exercise-induced hyperglycemia and returned BG to that of a non-exercise trial.

Reductions in resistance exercise-induced hyperglycaemic episodes are associated with circulating interleukin-6 in type 1 diabetes.

AIMS: To determine the influence of different volumes of resistance exercise on circulating interleukin-6 (IL-6) and to explore the relationships between IL-6 and glycaemia. METHODS: Eight participants with complication-free type 1 diabetes, whose mean ± SEM age was 38 (6) years, mean ± SEM HbA(1c) concentration was 71 ±11 mmol/mol (8.7 ±1.0%) and mean ± SEM type 1 diabetes duration was 15 ±13 years, attended the research facility after an overnight fast on four separate occasions, having administered their basal insulin the night before (glargine 27.5±3.1U, n=8), but omitted morning rapid-acting insulin. Participants completed either a one-set (14-min), two-set (28-min), or three-set (42-min) resistance exercise trial (eight exercises × 10 repetitions) at 67±3% one-repetition maximum followed by a 60-min recovery, or a resting control trial. Venous blood samples were taken before and after exercise. Data were analysed using repeated-measures ANOVA (P≤0.05). RESULTS: Whereas IL-6 levels remained similar to baseline levels after one set of resistance exercises (30 min, P=0.287; 60 min, P=0.318), IL-6 levels were > baseline levels at 60 min post-exercise after a two-set exercise trial (2.94 ± 0.94 pg/ml, P=0.002) and doubled at both 30 min (4.01 ± 1.00 pg/ml, P=0.048) and 60 min (4.28 ± 1.25 pg/ml, P=0.084) post-exercise after the three-set resistance exercise trial. Post-exercise blood glucose area under the curve (mmol/l/60 min) was greater after both the one-set (P=0.025) and two-set trials (P=0.008), than after the control trial, but similar between the three-set trial and the control trial (P=0.240). The rise in IL-6 from baseline to peak concentration significantly correlated inversely with blood glucose area under the curve (r=-0.65, P=0.041). CONCLUSIONS: Circulating IL-6 is increased by resistance exercise in a volume-dependent manner, and resistance exercise-induced increases in IL-6 correlated with reductions in post-exercise hyperglycaemia in type 1 diabetes, suggesting a role for IL-6 in improving post-resistance exercise glycaemic disturbances in type 1 diabetes.

The influence of calcium supplementation on substrate metabolism during exercise in humans: a randomized controlled trial.

BACKGROUND/OBJECTIVES: High calcium intakes enhance fat loss under restricted energy intake. Mechanisms explaining this may involve reduced dietary fat absorption, enhanced lipid utilization and (or) reductions in appetite. This study aimed to assess the impact of 2 weeks of calcium supplementation on substrate utilization during exercise and appetite sensations at rest. SUBJECTS/METHODS: Thirteen physically active males completed two 14-d supplemental periods, in a double-blind, randomized crossover design separated by a ⩾4-week washout period. During supplementation, a test-drink was consumed daily containing 400 and 1400 mg of calcium during control (CON) and high-calcium (CAL) periods, respectively. Cycling-based exercise tests were conducted before and after each supplemental period to determine substrate utilization rates and circulating metabolic markers (non-esterified fatty acid, glycerol, glucose and lactate concentrations) across a range of exercise intensities. Visual analog scales were completed in the fasting, rested state to determine subjective appetite sensations. RESULTS: No significant differences between supplements were observed in lipid or carbohydrate utilization rates, nor in circulating metabolic markers (both P>0.05). Maximum rates of lipid utilization were 0.47±0.05 and 0.44±0.05 g/min for CON and CAL, respectively, prior to supplementation and 0.44±0.05 and 0.42±0.05 g/min, respectively, post-supplementation (main effects of time, supplement and time x supplement interaction effect all P>0.05). Furthermore, no significant differences were detected in any subjective appetite sensations (all P>0.05). CONCLUSIONS: Two weeks of calcium supplementation does not influence substrate utilization during exercise in physically active males.

Development and use of real-time PCR to detect and quantify Mycoplasma haemocanis and "Candidatus Mycoplasma haematoparvum" in dogs.

Two canine haemoplasma species have been recognised to date; Mycoplasma haemocanis (Mhc), which has been associated with anaemia in splenectomised or immunocompromised dogs, and "Candidatus Mycoplasma haematoparvum" (CMhp), recently described in an anaemic splenectomised dog undergoing chemotherapy. The study aim was to develop quantitative real-time PCR assays (qPCRs) incorporating an endogenous internal control to detect Mhc and CMhp and to apply these assays to DNA samples extracted from canine blood collected in Northern Tanzania (n=100) and from dogs presented to a Trinidadian veterinary hospital (n=185). QPCRs specific for Mhc and CMhp were designed using 16S rRNA gene sequence data, and each was duplexed with an assay specific for canine glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The assays detected < or =10 copies of a sequence-specific haemoplasma plasmid per reaction and neither assay showed cross-reactivity with 10(6) copies of the sequence-specific plasmid from the non-target canine haemoplasma species. Nineteen of the 100 Tanzanian samples (19%) were positive for Mhc alone and one (1%) was dually infected. One Trinidadian sample was negative for canine GAPDH DNA and was excluded from the study. Of the 184 remaining Trinidadian samples, nine (4.9%) were positive for Mhc alone, five (2.7%) for CMhp alone, and two (1.1%) dually infected. This is the first report of canine haemoplasma qPCR assays that use an internal control to confirm the presence of amplifiable sample DNA, and their application to prevalence studies. Mhc was the most commonly detected canine haemoplasma species.

Chromomycosis in a dog due to Torula sp[ecies]

Post-mortem examination of a mixed breed dog, showing severe neurological signs, revealed brownish-black nodular lesions of the liver, kidney, heart, lungs and brain. Histologically, the lesions revealed numerous dark-brown fungal elements suggestive of a mycotic infection. The isolate was identified as Torula sp. (AU)

Endocardial ossifying myxoma of the right atrium in a cat.

Primary cardiac neoplasms are rare in humans and animals. In humans, the most common primary cardiac tumor is the myxoma, which is frequently found in the left atrium. Cardiac myxoma has been reported in the dog but not in the cat. We describe the gross, immunohistochemical, and light microscopic examination of a myxoma in the right atrium of a 6-year-old domestic shorthair cat. Histologically, the tumor consisted predominantly of mesenchymal cells with several foci of bone and cartilage present. The tumor was encapsulated and benign.

The fall, recovery, orbit, and composition of the Tagish Lake meteorite: a new type of carbonaceous chondrite.

The preatmospheric mass of the Tagish Lake meteoroid was about 200,000 kilograms. Its calculated orbit indicates affinity to the Apollo asteroids with a semimajor axis in the middle of the asteroid belt, consistent with a linkage to low-albedo C, D, and P type asteroids. The mineralogy, oxygen isotope, and bulk chemical composition of recovered samples of the Tagish Lake meteorite are intermediate between CM and CI meteorites. These data suggest that the Tagish Lake meteorite may be one of the most primitive solar system materials yet studied.

Polyarticular blastomycotic arthritis.

Blastomycotic arthritis usually presents as acute monoarticular septic arthritis resembling a bacterial process. Only 2 cases in the literature have subsequently developed arthritis in a second joint. We describe the case of an elderly woman with polyarticular blastomycotic arthritis associated with lung, skin, and bone involvement. The organism was identified in the synovial fluid and skin biopsies. Our patient underwent open drainage and aspiration of the involved joints and received antimycotic therapy.

Femoral component failure in hybrid total knee arthroplasty.

From 1986 to 1991, 74 consecutive hybrid total knee arthroplasties (in 65 patients) of a single design were performed at the authors' institution. Seven patients (eight knees) died with well functioning replacements during the surveillance period. One patient refused to participate in any followup efforts because of billing disputes. This left 65 total knee arthroplasties (57 patients) for review. There were 35 men and 22 women with an average age of 60 years (range, 27-83 years) at the time of arthroplasty. The underlying diagnosis was osteoarthritis in 46 knees, rheumatoid arthritis in six knees, and other in four knees. The average length of followup was 7.4 years (range, 5-10 years), and no patients were lost to followup. The Knee Society scores improved from an average of 37 preoperatively to 84 at latest followup. The functional score improved from 49 to 69 at latest followup. Ten (13.8%) knees required revision surgery. Eight of the nine knees that were revised had femoral component problems, including a loose component in six and a fractured component in two. One knee was revised for polyethylene wear with secondary osteolysis and one was revised elsewhere in which the operative details are unknown. The implant survival to revision at 5 years was 89% and 85% at final followup. Femoral component fixation in hybrid total knee arthroplasty in unreliable with this component design. In light of the excellent 10- to 15-year results of cemented condylar knee designs it is thought that hybrid fixation should be abandoned.

Free radical-induced generation of isoprostanes in vivo. Evidence for the formation of D-ring and E-ring isoprostanes.

We recently reported the discovery that a series of novel prostaglandin (PG)F2-like compounds (F2-isoprostanes) are produced in vivo independent of the cyclooxygenase as products of free radical-catalyzed lipid peroxidation. F2-isoprostanes are initially formed in situ from arachidonic acid esterified to phospholipids and then released preformed. We have now investigated whether PGD2/E2-like isoprostanes are also produced by rearrangement of the PGG2-like intermediates involved in isoprostane formation. Using a variety of approaches utilizing mass spectrometry, compelling evidence was obtained for the presence of D2/E2-isoprostane-containing phosphospholipids in the liver (85 +/- 33 ng/g of liver) and free D2/E2-isoprostanes in the circulation (215 +/- 90 pg/ml) of rats treated with CCl4 to induce lipid peroxidation. In untreated rats, levels of D2/E2-isoprostanes esterified in liver phospholipids were much lower (0.90 +/- 0.10 ng/g), and free compounds could not be detected in the circulation (< 5 pg/ml). Interestingly, one of the E2-isoprostanes that would be expected to be formed in abundance, 8-epi-PGE2, was found to be a potent renal vasoconstrictor, and these effects could be abrogated by SQ29548, a thromboxane receptor antagonist. Further understanding of the biological consequences of the formation of these novel compounds and factors that influence their formation may provide valuable insights into the pathophysiology of oxidant injury.

Cyclic AMP inhibits inositol polyphosphate production and calcium mobilization in neuroblastoma X glioma NG108-15 cells.

In the neuroblastoma X glioma hybrid cell line NG108-15, bradykinin (BK) receptor stimulation induced a rapid and concentration-dependent rise in cytosolic free Ca2+ levels, as measured with the Ca2(+)-sensitive fluorescent dye fura-2. The Ca2+ transient was present in the absence of extracellular Ca2+ and was associated with a concentration-dependent production of inositol phosphates, particularly inositol trisphosphate (InsP3). Pretreatment of intact NG108-15 cells with forskolin or dibutyryl-cAMP plus isobutylmethylxanthine reduced BK-stimulated InsP3 production and the increase in cytosolic free Ca2+. Membranes prepared from forskolin- and [3H]inositol-pretreated NG108-15 cells also showed a diminished production of InsP3 elicited by guanosine 5'-[gamma-thio]triphosphate, NaF, or BK plus GTP. On the other hand, the Ca2+ sensitivity of membrane-associated phosphoinositide-specific phospholipase C (PI-PLC) was unaffected by forskolin pretreatment of intact NG108-15 cells. Collectively, these results suggest that A-kinase may inhibit receptor-mediated and postreceptor stimulation of PI-PLC in neuron-like cells, perhaps by impairing the coupling between a guanine nucleotide-binding protein and PI-PLC.

Reduction of norepinephrine-induced tonic contraction and phosphoinositide turnover in arteries of spontaneously hypertensive rats. A possible role for protein kinase C.

Experiments were conducted to determine whether a difference in receptor-induced phosphatidylinositol hydrolysis occurred in aorta from spontaneously hypertensive rats (SHR) v Wistar-Kyoto (WKY) rats, and whether such a difference was correlated with contractile response. Basal incorporation of 32P into phosphatidylinositol (PI), phosphatidylinositol phosphate (PIP), phosphatidylinositol diphosphate (PIP2) and phosphatidic-acid (PA) was not different between SHR and WKY groups. However, after five minutes of norepinephrine (NE; 10 mumol) exposure, increases in 32P labeling were markedly lower in SHR arteries. The percentage decrease amounted to 45% for PI, 68% for PIP, 100% for PIP2 and 58% for PA. Basal incorporation of 3H-myo-inositol into inositol monophosphate (IP) was similar for SHR and WKY groups. However, after 30 minutes of NE (10 mumol), SHR arteries failed to show an increase in 3H-IP levels, whereas labeling was increased 219% in WKY arteries. The contractile response of SHR arteries to 10 mumol NE showed a marked reduction in the rate of development of the tonic phase that has previously been shown to be supported by activity of protein kinase C. Higher Ca2+ levels failed to augment the SHR response, whereas WKY responses were significantly increased. Contractions in the presence of the phorbol ester tetradecanoylphorbolacetate exhibited a similar reduction in NE-induced tonic phase tension. These results indicate an impairment in SHR arteries at the level of receptor-induced formation of inositol cycle second messengers, possibly due to elevated basal levels of protein kinase C. These differences may be important in explaining altered vascular responses in primary hypertension.