Tyrosine hydroxylase immunoreactivity as indicator of sympathetic activity: simultaneous evaluation in different tissues of hypertensive rats

Burgi K, Cavalleri MT, Alves AS, Britto LRG, Antunes VR, Michelini LC. Tyrosine hydroxylase immunoreactivity as indicator of sympathetic activity: simultaneous evaluation in different tissues of hypertensive rats. Am J Physiol Regul Integr Comp Physiol 300: R264-R271, 2011. First published December 9, 2010; doi: 10.1152/ajpregu.00687.2009.-Vasomotor control by the sympathetic nervous system presents substantial heterogeneity within different tissues, providing appropriate homeostatic responses to maintain basal/stimulated cardiovascular function both at normal and pathological conditions. The availability of a reproducible technique for simultaneous measurement of sympathetic drive to different tissues is of great interest to uncover regional patterns of sympathetic nerve activity (SNA). We propose the association of tyrosine hydroxylase immunoreactivity (THir) with image analysis to quantify norepinephrine (NE) content within nerve terminals in arteries/arterioles as a good index for regional sympathetic outflow. THir was measured in fixed arterioles of kidney, heart, and skeletal muscle of WistarKyoto rats (WKY) and spontaneously hypertensive rats (SHR) (123 +/- 2 and 181 +/- 4 mmHg, 300 +/- 8 and 352 +/- 8 beats/min, respectively). There was a differential THir distribution in both groups: higher THir was observed in the kidney and skeletal muscle (similar to 3-4-fold vs. heart arterioles) of WKY; in SHR, THir was increased in the kidney and heart (2.4- and 5.3-fold vs. WKY, respectively) with no change in the skeletal muscle arterioles. Observed THir changes were confirmed by either: 1) determination of NE content (high-performance liquid chromatography) in fresh tissues (SHR vs. WKY): +34% and +17% in kidney and heart, respectively, with no change in the skeletal muscle; 2) direct recording of renal (RSNA) and lumbar SNA (LSNA) in anesthetized rats, showing increased RSNA but unchanged LSNA in SHR vs. WKY. THir in skeletal muscle arterioles, NE content in femoral artery, and LSNA were simultaneously reduced by exercise training in the WKY group. Results indicate that THir is a valuable technique to simultaneously evaluate regional patterns of sympathetic activity.

Neuromuscular electrical stimulation improves GLUT-4 and morphological characteristics of skeletal muscle in rats with heart failure

Aim: Changes in skeletal muscle morphology and metabolism are associated with limited functional capacity in heart failure, which can be attenuated by neuromuscular electrical stimulation (ES). The purpose of the present study was to analyse the effects of ES upon GLUT-4 protein content, fibre structure and vessel density of the skeletal muscle in a rat model of HF subsequent to myocardial infarction. Methods: Forty-four male Wistar rats were assigned to one of four groups: sham (S), sham submitted to ES (S+ES), heart failure (HF) and heart failure submitted to ES (HF+ES). The rats in the ES groups were submitted to ES of the left leg during 20 days (2.5 kHz, once a day, 30 min, duty cycle 50%- 15 s contraction/15 s rest). After this period, the left tibialis anterior muscle was collected from all the rats for analysis. Results: HF+ES rats showed lower values of lung congestion when compared with HF rats (P = 0.0001). Although muscle weight was lower in HF rats than in the S group, thus indicating hypotrophy, 20 days of ES led to their recovery (P < 0.0001). In both groups submitted to ES, there was an increase in muscle vessel density (P < 0.04). Additionally, heart failure determined a 49% reduction in GLUT-4 protein content (P < 0.03), which was recovered by ES (P < 0.01). Conclusion: In heart failure, ES improves morphological changes and raises GLUT-4 content in skeletal muscle.

UPR induces transient burst of apoptosis in islets of early lactating rats through reduced AKT phosphorylation via ATF4/CHOP stimulation of TRB3 expression

Bromati CR, Lellis-Santos C, Yamanaka TS, Nogueira TC, Leonelli M, Caperuto LC, Gorjao R, Leite AR, Anhe GF, Bordin S. UPR induces transient burst of apoptosis in islets of early lactating rats through reduced AKT phosphorylation via ATF4/CHOP stimulation of TRB3 expression. Am J Physiol Regul Integr Comp Physiol 300: R92-R100, 2011. First published November 10, 2010; doi:10.1152/ajpregu.00169.2010.-Endocrine pancreas from pregnant rats undergoes several adaptations that comprise increase in beta-cell number, mass and insulin secretion, and reduction of apoptosis. Lactogens are the main hormones that account for these changes. Maternal pancreas, however, returns to a nonpregnant state just after the delivery. The precise mechanism by which this reversal occurs is not settled but, in spite of high lactogen levels, a transient increase in apoptosis was already reported as early as the 3rd day of lactation (L3). Our results revealed that maternal islets displayed a transient increase in DNA fragmentation at L3, in parallel with decreased RAC-alpha serine/threonine-protein kinase (AKT) phosphorylation (pAKT), a known prosurvival kinase. Wortmannin completely abolished the prosurvival action of prolactin (PRL) in cultured islets. Decreased pAKT in L3-islets correlated with increased Tribble 3 (TRB3) expression, a pseudokinase inhibitor of AKT. PERK and eIF2 alpha phosphorylation transiently increased in islets from rats at the first day after delivery, followed by an increase in immunoglobulin heavy chain-binding protein (BiP), activating transcription factor 4 (ATF4), and C/EBP homologous protein (CHOP) in islets from L3 rats. Chromatin immunoprecipitation (ChIP) and Re-ChIP experiments further confirmed increased binding of the heterodimer ATF4/CHOP to the TRB3 promoter in L3 islets. Treatment with PBA, a chemical chaperone that inhibits UPR, restored pAKT levels and inhibited the increase in apoptosis found in L3. Moreover, PBA reduced CHOP and TRB3 levels in beta-cell from L3 rats. Altogether, our study collects compelling evidence that UPR underlies the physiological and transient increase in beta-cell apoptosis after delivery. The UPR is likely to counteract prosurvival actions of PRL by reducing pAKT through ATF4/CHOP-induced TRB3 expression.

SGLT1 protein expression in plasma membrane of acinar cells correlates with the sympathetic outflow to salivary glands in diabetic and hypertensive rats

Salivary gland dysfunction is a feature in diabetes and hypertension. We hypothesized that sodium-glucose cotransporter 1 (SGLT1) participates in salivary dysfunctions through a sympathetic- and protein kinase A (PKA)-mediated pathway. In Wistar-Kyoto (WKY), diabetic WKY (WKY-D), spontaneously hypertensive (SHR), and diabetic SHR (SHR-D) rats, PKA/SGLT1 proteins were analyzed in parotid and submandibular glands, and the sympathetic nerve activity (SNA) to the glands was monitored. Basal SNA was threefold higher in SHR (P < 0.001 vs. WKY), and diabetes decreased this activity (similar to 50%, P < 0.05) in both WKY and SHR. The catalytic subunit of PKA and the plasma membrane SGLT1 content in acinar cells were regulated in parallel to the SNA. Electrical stimulation of the sympathetic branch to salivary glands increased (similar to 30%, P < 0.05) PKA and SGLT1 expression. Immunohistochemical analysis confirmed the observed regulations of SGLT1, revealing its location in basolateral membrane of acinar cells. Taken together, our results show highly coordinated regulation of sympathetic activity upon PKA activity and plasma membrane SGLT1 content in salivary glands. Furthermore, the present findings show that diabetic- and/or hypertensive-induced changes in the sympathetic activity correlate with changes in SGLT1 expression in basolateral membrane of acinar cells, which can participate in the salivary glands dysfunctions reported by patients with these pathologies.

Interaction with calmodulin is important for the secretion of thimet oligopeptidase following stimulation

Thimet oligopeptidase (EC 3.4.24.15; EP24.15) was originally described as a neuropeptide-metabolizing enzyme, highly expressed in the brain, kidneys and neuroendocrine tissue. EP24.15 lacks a typical signal peptide sequence for entry into the secretory pathway and is secreted by cells via an unconventional and unknown mechanism. In this study, we identified a novel calcium-dependent interaction between EP24.15 and calmodulin, which is important for the stimulated, but not constitutive, secretion of EP24.15. We demonstrated that, in vitro, EP24.15 and calmodulin physically interact only in the presence of Ca(2+), with an estimated K(d) value of 0.52 mu m. Confocal microscopy confirmed that EP24.15 colocalizes with calmodulin in the cytosol of resting HEK293 cells. This colocalization markedly increases when cells are treated with either the calcium ionophore A23187 or the protein kinase A activator forskolin. Overexpression of calmodulin in HEK293 cells is sufficient to greatly increase the A23187-stimulated secretion of EP24.15, which can be inhibited by the calmodulin inhibitor calmidazolium. The specific inhibition of protein kinase A with KT5720 reduces the A23187-stimulated secretion of EP24.15 and inhibits the synergistic effects of forskolin with A23187. Treatment with calmidazolium and KT5720 nearly abolishes the stimulatory effects of A23187 on EP24.15 secretion. Together, these data suggest that the interaction between EP24.15 and calmodulin is regulated within cells and is important for the stimulated secretion of EP24.15 from HEK293 cells.

Matrix metalloproteinases cleave the beta(2)-adrenergic receptor in spontaneously hypertensive rats

Rodrigues SF, Tran ED, Fortes ZB, Schmid-Schonbein GW. Matrix metalloproteinases cleave the beta(2)-adrenergic receptor in spontaneously hypertensive rats. Am J Physiol Heart Circ Physiol 299: H25-H35, 2010. First published April 9, 2010; doi:10.1152/ajpheart.00620.2009.-We recently observed the enhanced serine and matrix metalloproteinase (MMP) activity in the spontaneously hypertensive rat (SHR) compared with its normotensive Wistar-Kyoto (WKY) rat and the cleavage of membrane receptors in the SHR by MMPs. We demonstrate in vivo that MMP-7 and MMP-9 injection leads to a vasoconstrictor response in microvessels of rats that is blocked by a specific MMP inhibitor (GM-6001, 1 mu M). Multiple pathways may be responsible. Since the beta(2)-adrenergic receptor (beta(2)-AR) is susceptible to the action of endogenous MMPs, we hypothesize that MMPs in the plasma of SHRs are able to cleave the extracellular domain of the beta(2)-AR. SHR arterioles respond in an attenuated fashion to beta(2)-AR agonists and antagonists. Aorta and heart muscle of control Wistar rats were exposed for 24 h (37 C) to fresh plasma of male Wistar and WKY rats and SHRs with and without doxycycline (30 mu M) and EDTA (10 mM) to reduce MMP activity. The density of extracellular and intracellular domains of beta(2)-AR was determined by immunohistochemistry. The density of the extracellular domain of beta(2)-AR is reduced in aortic endothelial cells and cardiac microvessels of SHRs compared with that of WKY or Wistar rats. Treatment of the aorta and the heart of control Wistar rats with plasma from SHRs, but not from WKY rats, reduced the number of extracellular domains, but not intracellular domains, of beta(2)-AR in aortic endothelial cells and cardiac microvessels. MMP inhibitors (EDTA and doxycycline) prevented the cleavage of the extracellular domain. Thus MMPs may contribute to the reduced density of the extracellular domain of beta(2)-AR in blood vessels and to the increased arteriolar tone of SHRs compared with normotensive rats.

Pyk2 mediates increased adrenergic contractile responses in arteries from DOCA-salt mice - Vasoactive Peptide Symposium

The calcium-dependent proline-rich tyrosine kinase (Pyk2), a nonreceptor protein activated by tyrosine phosphorylation, links G protein-coupled receptors to vascular responses. We tested the hypothesis that enhanced vascular reactivity in deoxycorticosterone acetate (DOCA)-salt hypertensive mice is due to increased activation of Pyk2. Aorta and small mesenteric arteries from DOCA-salt and uninephrectomized (UNI) male C57B1/6 mice were used. Systolic blood pressure (mm Hg) was higher in DOCA (126 +/- 3) vs. UNI (100 +/- 4) mice. Vascular responses to phenylephrine (1 nM to 100 mu M) were greater both in aorta and small mesenteric arteries from DOCA-salt than UNI, but treatment with Tyrphostin A-9 (0.1 mu M, Pyk2 inhibitor) abolished the difference among the groups. Pyk2 levels, as well as phospho-Pyk2(Tyr402), paxillin, and phospho-paxillin(Tyr118) were increased in DOCA-salt aorta. Incubation of vessels with Tyrphostin A-9 restored phosphorylation of Pyk2 and paxillin. Increased activation of Pyk2 contributes to increased vascular contractile responses in DOCA-salt mice. J Am Soc Hypertens 2008;2(6): 431-438. (C) 2008 American Society of Hypertension. All rights reserved.

Effect of neuromuscular electrical stimulation and isotonic exercises in flexor and extensor muscles of knee of hemiplegic patients

Aim. To verify the muscular force and resistance to the movement of the flexor and extensor muscles of the knee of patients with spasticity after treatment with neuromuscular electrical stimulation (NMES) and isotonic exercises. Patients and methods. The patients this study were divided into group I (NMES) and group 2 (isotonic exercises). Their muscular torque and resistance to the movement of the flexor and extensor knee muscles were measured by the isokinetic dynamometer and the degree of spasticity by the modified Ashworth scale before and after ten sessions. Results. Alterations in the scores of the modified Ashworth scale were not observed. An increase in the flexor torque in group 1 (p = 0.041) and in group 2 (p = 0.001) was verified. In the passive mode, group 1 presented a reduction of resistance to the flexion movement (p = 0.026), while in group 2, a reduction of resistance to both the flexion (p = 0,029) and extension movements (p = 0.019) was verified. Conclusions. The two therapeutical resources had their efficiency proven only for the increase of the force of the flexor muscles. The resistance to movement, the isotonic exercises were more effective because they promoted a reduction in the resistance of the flexor and extensor knee muscles.

Insulin temporal sensitivity and its signaling pathway in the rat pineal gland

Aims: In our previous work, we reported that the insulin potentiating effect on melatonin synthesis is regulated by a post-transcriptional mechanism. However, the major proteins of the insulin signaling pathway (ISP) and the possible pathway component recruited on the potentiating effect of insulin had not been characterized. A second question raised was whether windows of sensitivity to insulin exist in the pineal gland due to insulin rhythmic secretion pattern. Main methods: Melatonin content from norepinephrine(NE)-synchronized pineal gland cultures was quantified by high performance liquid chromatography with electrochemical detection and arylalkylamine-N-acetyltransferase (AANAT) activity was assayed by radiometry. Immunoblotting and immunoprecipitation techniques were performed to establish the ISP proteins expression and the formation of 14-3-3: AANAT complex, respectively. Key findings: The temporal insulin susceptibility protocol revealed two periods of insulin potentiating effect, one at the beginning and another one at the end of the in vitro induced ""night"". In some Timed-insulin Stimulation (TSs), insulin also promoted a reduction on melatonin synthesis, showing its dual action in cultured pineal glands. The major ISP components, such as IR beta, IGF-1R, IRS-1, IRS-2 and PI3K(p85), as well tyrosine phosphorylation of pp85 were characterized within pineal glands. Insulin is not involved in the 14-3-3:AANAT complex formation. The blockage of PI3K by LY 294002 reduced melatonin synthesis and AANAT activity. Significance: The present study demonstrated windows of differential insulin sensitivity, a functional ISP and the PI3K-dependent insulin potentiating effect on NE-mediated melatonin synthesis, supporting the hypothesis of a crosstalk between noradrenergic and insulin pathways in the rat pineal gland. (C) 2010 Elsevier Inc. All rights reserved.

Stimulation of Acidic Reduction of Nitrite to Nitric Oxide by Soybean Phenolics: Possible Relevance to Gastrointestinal Host Defense

This study aimed to evaluate the potential of soybean-promoted acidic nitrite reduction and to correlate this activity with the content of phenolics and with the bactericidal activity against Escherichia coli O157:H7. Extracts of embrionary axes and cotyledons enriched in phenolics increased (center dot)NO formation at acidic pH at values that were 7.1 and 4.5 times higher, respectively, when compared to the reduction of the nonenriched extracts. Among the various phenolics accumulated in the soybean extracts, five stimulated nitrite reduction in the following decreasing order of potency: epicatechin gallate, chlorogenic acid, caffeic acid, galic acid and p-coumaric acid. Extracts of embrionary axes presented higher contents of epicatechin gallate and caffeic acid, compared to that of cotyledons, indicating a positive correlation between activity of the extracts and content of phenolics with regard to nitrite reducing activity. Soybean extracts enriched in phenolics interacted synergistically with acidified nitrite to prevent E. coli O157:H7 growth. The results suggest that soybean phenolics may interfere with the metabolism of (center dot)NO in an acidic environment by accelerating the reduction of nitrite, with a potential antimicrobial effect in the stomach.

Acupuncture with manual and electrical stimulation for labour pain : a longitudinal randomised controlled trial

Background: Acupuncture is commonly used to reduce pain during labour despite contradictory results. The aim of this study is to evaluate the effectiveness of acupuncture with manual stimulation and acupuncture with combined manual and electrical stimulation (electro-acupuncture) compared with standard care in reducing labour pain. Our hypothesis was that both acupuncture stimulation techniques were more effective than standard care, and that electro-acupuncture was most effective.  Methods: A longitudinal randomised controlled trial. The recruitment of participants took place at the admission to the labour ward between November 2008 and October 2011 at two Swedish hospitals. 303 nulliparous women with normal pregnancies were randomised to: 40 minutes of manual acupuncture (MA), electro-acupuncture (EA), or standard care without acupuncture (SC). Primary outcome: labour pain, assessed by Visual Analogue Scale (VAS). Secondary outcomes: relaxation, use of obstetric pain relief during labour and post-partum assessments of labour pain. The sample size calculation was based on the primary outcome and a difference of 15 mm on VAS was regarded as clinically relevant, this gave 101 in each group, including a total of 303 women.  Results: Mean estimated pain scores on VAS (SC: 69.0, MA: 66.4 and EA: 68.5), adjusted for: treatment, age, education, and time from baseline, with no interactions did not differ between the groups (SC vs MA: mean difference 2.6, 95% confidence interval [CI] -1.7-6.9 and SC vs EA: mean difference 0.6 [95% CI] -3.6-4.8). Fewer number of women in the EA group used epidural analgesia (46%) than women in the MA group (61%) and SC group (70%) (EA vs SC: odds ratio [OR] 0.35; [95% CI] 0.19-0.67).  Conclusions: Acupuncture does not reduce women's experience of labour pain, neither with manual stimulation nor with combined manual and electrical stimulation. However, fewer women in the EA group used epidural analgesia thus indicating that the effect of acupuncture with electrical stimulation may be underestimated. These findings were obtained in a context with free access to other forms of pain relief.

Acupuncture with manual and electrical stimulation for labour pain : a two month follow up of recollection of pain and birth experience.

BACKGROUND: In a previous randomised controlled trial we showed that acupuncture with a combination of manual- and electrical stimulation (EA) did not affect the level of pain, as compared with acupuncture with manual stimulation (MA) and standard care (SC), but reduced the need for other forms of pain relief, including epidural analgesia. To dismiss an under-treatment of pain in the trial, we did a long-term follow up on the recollection of labour pain and the birth experience comparing acupuncture with manual stimulation, acupuncture with combined electrical and manual stimulation with standard care. Our hypothesis was that despite the lower frequency of use of other pain relief, women who had received EA would make similar retrospective assessments of labour pain and the birth experience 2 months after birth as women who received standard care (SC) or acupuncture with manual stimulation (MA). METHODS: Secondary analyses of data collected for a randomised controlled trial conducted at two delivery wards in Sweden. A total of 303 nulliparous women with normal pregnancies were randomised to: 40 min of MA or EA, or SC without acupuncture. Questionnaires were administered the day after partus and 2 months later. RESULTS: Two months postpartum, the mean recalled pain on the visual analogue scale (SC: 70.1, MA: 69.3 and EA: 68.7) did not differ between the groups (SC vs MA: adjusted mean difference 0.8, 95 % confidence interval [CI] -6.3 to 7.9 and SC vs EA: mean difference 1.3 CI 95 % -5.5 to 8.1). Positive birth experience (SC: 54.3 %, MA: 64.6 % and EA: 61.0 %) did not differ between the groups (SC vs MA: adjusted Odds Ratio [OR] 1.8, CI 95 % 0.9 to 3.7 and SC vs EA: OR 1.4 CI 95 % 0.7 to 2.6). CONCLUSIONS: Despite the lower use of other pain relief, women who received acupuncture with the combination of manual and electrical stimulation during labour made the same retrospective assessments of labour pain and birth experience 2 months postpartum as those who received acupuncture with manual stimulation or standard care. TRIAL REGISTRATION: ClinicalTrials.gov: NCT01197950.

The Pair-Bond Formation and Its Role in the Stimulation of Reproductive Function in Female Common Marmosets (Callithrix jacchus)

SILVA, H.P.A.; SOUSA, M.B.C. The pair-bond formation and its role in the stimulation of reproductive function in female common marmosets (collithrix Jacchus). International Journal of Primatology, v, 18, n.3, p.387-400, 1997.

Cardiovascular changes under normoxic and hypoxic conditions in the air-breathing teleost Synbranchus marmoratus: importance of the venous system

Synbranchus marmoratus is a facultative air-breathing fish, which uses its buccal cavity as well as its gills for air-breathing. S. marmoratus shows a very pronounced tachycardia when it surfaces to air-breathe. An elevation of heart rate decreases cardiac filling time and therefore may cause a decline in stroke volume (VS), but this can be compensated for by an increase in venous tone to maintain stroke volume. Thus, the study on S. marmoratus was undertaken to investigate how stroke volume and venous function are affected during air-breathing. To this end we measured cardiac output (Q), heart rate (fH), central venous blood pressure (PCV), mean circulatory filling pressure (MCFP), and dorsal aortic blood pressures (PDA) in S. marmoratus. Measurements were performed in aerated water (P-O2 > 130 mmHg), when the fish alternated between gill ventilation and prolonged periods of apnoeas, as well as during hypoxia (P-O2 <= 50 mmHg), when the fish changed from gill ventilation to air-breathing. Q increased significantly during gill ventilation compared to apnoea in aerated water through a significant increase in both fH and VS. PCV and MCFP also increased significantly. During hypoxia, when the animals surface to ventilate air, we found a marked rise in fH, PCV, MCFP, Q and VS, whereas PDA decreased significantly. Simultaneous increases in PCV and MCFP in aerated, as well as in hypoxic water, suggests that the venous system plays an important regulatory role for cardiac filling and VS in this species. In addition, we investigated adrenergic regulation of the venous system through bolus infusions of adrenergic agonists (adrenaline, phenylephrine and isoproterenol; 2 mu g kg(-1)). Adrenaline and phenylephrine caused a marked rise in PCV and MCFP, whereas isoproterenol led to a marked decrease in PCV, and tended to decrease MCFP. Thus, it is evident that stimulation of both alpha- and beta-adrenoreceptors affects venous tone in S. marmoratus.