Resistance to thyroid hormone induced tachycardia in RTHα syndrome.

Mutations in thyroid hormone receptor α1 (TRα1) cause Resistance to Thyroid Hormone α (RTHα), a disorder characterized by hypothyroidism in TRα1-expressing tissues including the heart. Surprisingly, we report that treatment of RTHα patients with thyroxine to overcome tissue hormone resistance does not elevate their heart rate. Cardiac telemetry in male, TRα1 mutant, mice indicates that such persistent bradycardia is caused by an intrinsic cardiac defect and not due to altered autonomic control. Transcriptomic analyses show preserved, thyroid hormone (T3)-dependent upregulation of pacemaker channels (Hcn2, Hcn4), but irreversibly reduced expression of several ion channel genes controlling heart rate. Exposure of TRα1 mutant male mice to higher maternal T3 concentrations in utero, restores altered expression and DNA methylation of ion channels, including Ryr2. Our findings indicate that target genes other than Hcn2 and Hcn4 mediate T3-induced tachycardia and suggest that treatment of RTHα patients with thyroxine in high dosage without concomitant tachycardia, is possible.

Development and prevention of ischemic contracture ("stone heart") in the pig heart.

Stone heart (ischemic contracture) is a rare and serious condition observed in the heart after periods of warm ischemia. The underlying mechanisms are largely unknown and treatment options are lacking. In view of the possibilities for cardiac donation after circulatory death (DCD), introducing risks for ischemic damage, we have investigated stone heart in pigs. Following cessation of ventilation, circulatory death (systolic pressure <8 mmHg) occurred within 13.1 ± 1.2 min; and a stone heart, manifested with asystole, increased left ventricular wall thickness and stiffness, established after a further 17 ± 6 min. Adenosine triphosphate and phosphocreatine levels decreased by about 50% in the stone heart. Electron microscopy showed deteriorated structure with contraction bands, Z-line streaming and swollen mitochondria. Synchrotron based small angle X-ray scattering of trabecular samples from stone hearts revealed attachment of myosin to actin, without volume changes in the sarcomeres. Ca2+ sensitivity, determined in permeabilized muscle, was increased in stone heart samples. An in vitro model for stone heart, using isolated trabecular muscle exposed to hypoxia/zero glucose, exhibited the main characteristics of stone heart in whole animals, with a fall in high-energy phosphates and development of muscle contracture. The stone heart condition in vitro was significantly attenuated by the myosin inhibitor MYK-461 (Mavacamten). In conclusion, the stone heart is a hypercontracted state associated with myosin binding to actin and increased Ca2+ sensitivity. The hypercontractile state, once developed, is poorly reversible. The myosin inhibitor MYK-461, which is clinically approved for other indications, could be a promising venue for prevention.

A small molecule inhibitor of Nox2 and Nox4 improves contractile function after ischemia-reperfusion in the mouse heart.

The NADPH oxidase enzymes Nox2 and 4, are important generators of Reactive oxygen species (ROS). These enzymes are abundantly expressed in cardiomyocytes and have been implicated in ischemia-reperfusion injury. Previous attempts with full inhibition of their activity using genetically modified animals have shown variable results, suggesting that a selective and graded inhibition could be a more relevant approach. We have, using chemical library screening, identified a new compound (GLX481304) which inhibits Nox 2 and 4 (with IC50 values of 1.25 µM) without general antioxidant effects or inhibitory effects on Nox 1. The compound inhibits ROS production in isolated mouse cardiomyocytes and improves cardiomyocyte contractility and contraction of whole retrogradely (Langendorff) perfused hearts after a global ischemia period. We conclude that a pharmacological and partial inhibition of ROS production by inhibition of Nox 2 and 4 is beneficial for recovery after ischemia reperfusion and might be a promising venue for treatment of ischemic injury to the heart.

In Vivo Function of the Chaperonin TRiC in α-Actin Folding during Sarcomere Assembly.

The TCP-1 ring complex (TRiC) is a multi-subunit group II chaperonin that assists nascent or misfolded proteins to attain their native conformation in an ATP-dependent manner. Functional studies in yeast have suggested that TRiC is an essential and generalized component of the protein-folding machinery of eukaryotic cells. However, TRiC's involvement in specific cellular processes within multicellular organisms is largely unknown because little validation of TRiC function exists in animals. Our in vivo analysis reveals a surprisingly specific role of TRiC in the biogenesis of skeletal muscle α-actin during sarcomere assembly in myofibers. TRiC acts at the sarcomere's Z-disk, where it is required for efficient assembly of actin thin filaments. Binding of ATP specifically by the TRiC subunit Cct5 is required for efficient actin folding in vivo. Furthermore, mutant α-actin isoforms that result in nemaline myopathy in patients obtain their pathogenic conformation via this function of TRiC.

Long-term cardiovascular reprogramming by short-term perinatal exposure to nicotine's main metabolite cotinine.

AIM: Gather 'proof-of-concept' evidence of the adverse developmental potential of cotinine (a seemingly benign biomarker of recent nicotine/tobacco smoke exposure). METHODS: Pregnant C57 mice drank nicotine- or cotinine-laced water for 6 wks from conception (NPRE = 2% saccharin + 100 µg nicotine/mL; CPRE = 2% saccharin + 10 µg cotinine/mL) or 3 wks after birth (CPOST = 2% saccharin + 30 µg cotinine/mL). Controls drank 2% saccharin (CTRL). At 17 ± 1 weeks (male pups; CTRL n = 6; CPOST n = 6; CPRE n = 8; NPRE n = 9), we assessed (i) cardiovascular control during sleep; (ii) arterial reactivity ex vivo; and (iii) expression of genes involved in arterial constriction/dilation. RESULTS: Blood cotinine levels recapitulated those of passive smoker mothers' infants. Pups exposed to cotinine exhibited (i) mild bradycardia - hypotension at rest (p < 0.001); (ii) attenuated (CPRE , p < 0.0001) or reverse (CPOST ; p < 0.0001) BP stress reactivity; (iii) adrenergic hypocontractility (p < 0.0003), low protein kinase C (p < 0.001) and elevated adrenergic receptor mRNA (p < 0.05; all drug-treated arteries); and (iv) endothelial dysfunction (NPRE only). CONCLUSION: Cotinine has subtle, enduring developmental consequences. Some cardiovascular effects of nicotine can plausibly arise via conversion into cotinine. Low-level exposure to this metabolite may pose unrecognised perinatal risks. Adults must avoid inadvertently exposing a foetus or infant to cotinine as well as nicotine.

Inappropriate heat dissipation ignites brown fat thermogenesis in mice with a mutant thyroid hormone receptor α1.

Thyroid hormone is a major regulator of thermogenesis, acting both in peripheral organs and on central autonomic pathways. Mice heterozygous for a point mutation in thyroid hormone receptor α1 display increased thermogenesis as a consequence of high sympathetic brown fat stimulation. Surprisingly, despite the hypermetabolism, their body temperature is not elevated. Here we show, using isolated tail arteries, that defective thyroid hormone receptor α1 signaling impairs acetylcholine-mediated vascular relaxation as well as phenylephrine-induced vasoconstriction. Using infrared thermography on conscious animals, we demonstrate that these defects severely interfere with appropriate peripheral heat conservation and dissipation, which in turn leads to compensatory alterations in brown fat activity. Consequently, when the vasoconstrictive defect in mice heterozygous for a point mutation in thyroid hormone receptor α1 was reversed with the selective α1-adrenergic agonist midodrine, the inappropriate heat loss over their tail surface was reduced, normalizing brown fat activity and energy expenditure. Our analyses demonstrate that thyroid hormone plays a key role in vascular heat conservation and dissipation processes, adding a unique aspect to its well-documented functions in thermoregulation. The data thus facilitate understanding of temperature hypersensitivity in patients with thyroid disorders. Moreover, the previously unrecognized connection between cardiovascular regulation and metabolic activity revealed in this study challenges the interpretation of several experimental paradigms and questions some of the currently derived hypotheses on the role of thyroid hormone in thermogenesis.

Thyroid hormone is required for hypothalamic neurons regulating cardiovascular functions.

Thyroid hormone is well known for its profound direct effects on cardiovascular function and metabolism. Recent evidence, however, suggests that the hormone also regulates these systems indirectly through the central nervous system. While some of the molecular mechanisms underlying the hormone's central control of metabolism have been identified, its actions in the central cardiovascular control have remained enigmatic. Here, we describe a previously unknown population of parvalbuminergic neurons in the anterior hypothalamus that requires thyroid hormone receptor signaling for proper development. Specific stereotaxic ablation of these cells in the mouse resulted in hypertension and temperature-dependent tachycardia, indicating a role in the central autonomic control of blood pressure and heart rate. Moreover, the neurons exhibited intrinsic temperature sensitivity in patch-clamping experiments, providing a new connection between cardiovascular function and core temperature. Thus, the data identify what we believe to be a novel hypothalamic cell population potentially important for understanding hypertension and indicate developmental hypothyroidism as an epigenetic risk factor for cardiovascular disorders. Furthermore, the findings may be beneficial for treatment of the recently identified patients that have a mutation in thyroid hormone receptor α1.

AMP-activated kinase relaxes agonist induced contractions in the mouse aorta via effects on PKC signaling and inhibits NO-induced relaxation.

Adenosine monophosphate activated kinase (AMPK), a regulator of cellular metabolism, has been shown to relax arterial smooth muscle via endothelium-dependent and independent mechanisms. We have examined the role of AMPK in different smooth muscles using the activating compound, 5-amino-4-imidazolecarboxamide riboside-1-ß-d-ribofuranoside (AICAR). Isolated preparations of mouse aorta, saphenous artery, ileum and urinary bladder were compared. AICAR produced a reversible dose-dependent relaxation in aortic rings pre-incubated with AICAR and activated with phenylephrine. Less prominent relaxation was noted in the other tissues. This difference in sensitivity to AICAR was not due to differences in the expression levels of AMPK α1 mRNA. In the aorta, AICAR had a greater effect on contractions induced by phenylephrine, compared to high-K(+) induced contractions. Contractions of the aorta in response to the protein kinase C activator PDBu were prominently inhibited by AICAR. The AICAR relaxation observed in the aorta was not prevented by the NOS inhibitor L-NAME, Indomethacin or endothelium removal. Nitric oxide (NO) mediated relaxations in aortic preparations induced by acetylcholine or sodium nitroprusside (SNP) were attenuated by AICAR. In conclusion, AMPK induced relaxation of smooth muscle is tissue-dependent and most prominent in large elastic arteries. The smooth muscle relaxation is NO-independent and occurs downstream of PKC activation and is associated with attenuated relaxant responses to NO.

Busulphan-cyclophosphamide cause endothelial injury, remodeling of resistance arteries and enhanced expression of endothelial nitric oxide synthase.

Stem cell transplantation (SCT) is a curative treatment for malignant and non malignant diseases. However, transplantation-related complications including cardiovascular disease deteriorate the clinical outcome and quality of life. We have investigated the acute effects of conditioning regimen on the pharmacology, physiology and structure of large elastic arteries and small resistance-sized arteries in a SCT mouse model. Mesenteric resistance arteries and aorta were dissected from Balb/c mice conditioned with busulphan (Bu) and cyclophosphamide (Cy). In vitro isometric force development and pharmacology, in combination with RT-PCR, Western blotting and electron microscopy were used to study vascular properties. Compared with controls, mesenteric resistance arteries from the Bu-Cy group had larger internal circumference, showed enhanced endothelium mediated relaxation and increased expression of endothelial nitric oxide synthase (eNOS). Bu-Cy treated animals had lower mean blood pressure and signs of endothelial injury. Aortas of treated animals had a higher reactivity to noradrenaline. We conclude that short-term consequences of Bu-Cy treatment divergently affect large and small arteries of the cardiovascular system. The increased noradrenaline reactivity of large elastic arteries was not associated with increased blood pressure at rest. Instead, Bu-Cy treatment lowered blood pressure via augmented microvascular endothelial dependent relaxation, increased expression of vascular eNOS and remodeling toward a larger lumen. The changes in the properties of resistance arteries can be associated with direct effects of the compounds on vascular wall or possibly indirectly induced via altered translational activity associated with the reduced hematocrit and shear stress. This study contributes to understanding the mechanisms that underlie the early effects of conditioning regimen on resistance arteries and may help in designing further investigations to understand the late effects on vascular system.

Intimal hyperplasia in balloon dilated coronary arteries is reduced by local delivery of the NO donor, SIN-1 via a cGMP-dependent pathway.

BACKGROUND: To elucidate the mechanism by which local delivery of 3-morpholino-sydnonimine (SIN-1) affects intimal hyperplasia after percutaneous transluminal coronary angioplasty (PTCA). METHODS: Porcine coronary arteries were treated with PTCA and immediately afterwards locally treated for 5 minutes, with a selective cytosolic guanylate cyclase inhibitor, 1 H-(1,2,4)oxadiazole(4,3-alpha)quinoxaline-1-one (ODQ) + SIN-1 or only SIN-1 using a drug delivery-balloon. Arteries were angiographically depicted, morphologically evaluated and analyzed after one and eight weeks for actin, myosin and intermediate filaments (IF) and nitric oxide synthase (NOS) contents. RESULTS: Luminal diameter after PCI in arteries treated with SIN-1 alone and corrected for age-growth was significantly larger as compared to ODQ + SIN-1 or to controls (p < 0.01). IF/actin ratio after one week in SIN-1 treated segments was not different compared to untreated segments, but was significantly reduced compared to ODQ + SIN-1 treated vessels (p < 0.05). Expression of endothelial NADPH diaphorase activity was significantly lower in untreated segments and in SIN-1 treated segments compared to controls and SIN-1 + ODQ treated arteries (p < 0.01). Restenosis index (p < 0.01) and intimal hyperplasia (p < 0.01) were significantly reduced while the residual lumen was increased (p < 0.01) in SIN-1 segments compared to controls and ODQ + SIN-1 treated vessels. CONCLUSIONS: After PTCA local delivery of high concentrations of the NO donor SIN-1 for 5 minutes inhibited injury induced neointimal hyperplasia. This favorable effect was abolished by inhibition of guanylyl cyclase indicating mediation of a cyclic guanosine 3',5'-monophosphate (cGMP)-dependent pathway. The momentary events at the time of injury play crucial role in the ensuring development of intimal hyperplasia.

Multiple phenotypes in adult mice following inactivation of the Coxsackievirus and Adenovirus Receptor (Car) gene.

To determine the normal function of the Coxsackievirus and Adenovirus Receptor (CAR), a protein found in tight junctions and other intercellular complexes, we constructed a mouse line in which the CAR gene could be disrupted at any chosen time point in a broad spectrum of cell types and tissues. All knockouts examined displayed a dilated intestinal tract and atrophy of the exocrine pancreas with appearance of tubular complexes characteristic of acinar-to-ductal metaplasia. The mice also exhibited a complete atrio-ventricular block and abnormal thymopoiesis. These results demonstrate that CAR exerts important functions in the physiology of several organs in vivo.

Inflammation-induced airway smooth muscle responsiveness is strain dependent in mice.

Different mouse strains display different degrees of inflammation-induced airway hyperresponsiveness in vivo. It is not known whether these variations are attributable to distinct properties of the airway smooth muscle. Therefore, tracheal ring segments from C57BL/6 and BALB/c mice were exposed to three different pro-inflammatory stimuli for 4 days while maintained under tissue-culture conditions: tumour necrosis factor α (100 ng/ml), the Toll-like receptor (TLR) 3 agonist polyI:C (10 µg/ml), and the TLR4 agonist LPS (10 µg/ml). The contractile responses to carbachol, 5-hydroxytryptamine (5-HT) and bradykinin were assessed after culture. In addition, gene expression of TLR1-TLR9, pivotal inflammatory signal transduction proteins (jun-kinase, p38 and p65) and critical negative regulators of inflammation (A20, Itch, Tax1bp1 and RNF11) were studied in tracheal smooth muscle strips, fresh and following treatment for 4 days with LPS, from both strains. No differences between the strains were detected regarding the response of freshly isolated preparations to carbachol, 5-HT and bradykinin. After stimulation with pro-inflammatory mediators, contractions in response to 5-HT and bradykinin, but not to carbachol, were up-regulated. This up-regulation was markedly larger in BALB/c than in C57BL/6 segments and depended on the type of inflammatory stimulus. Expression of the genes investigated did not differ between the two strains. These findings indicate that strain differences in airway hyperresponsiveness can be linked to differences in the responsiveness of airway smooth muscle to pro-inflammatory mediators per se. The differences do not appear to be due to differential expression of TLR or common inflammatory transduction and repressor proteins.

Adaptations of the autonomous nervous system controlling heart rate are impaired by a mutant thyroid hormone receptor-alpha1.

Thyroid hormone has profound direct effects on cardiac function, but the hormonal interactions with the autonomic control of heart rate are unclear. Because thyroid hormone receptor (TR)-alpha1 has been implicated in the autonomic control of brown adipose energy metabolism, it might also play an important role in the central autonomic control of heart rate. Thus, we aimed to analyze the role of TRalpha1 signaling in the autonomic control of heart rate using an implantable radio telemetry system. We identified that mice expressing the mutant TRalpha1R384C (TRalpha1+m mice) displayed a mild bradycardia, which becomes more pronounced during night activity or on stress and is accompanied by a reduced expression of nucleotide-gated potassium channel 2 mRNA in the heart. Pharmacological blockage with scopolamine and the beta-adrenergic receptor antagonist timolol revealed that the autonomic control of cardiac activity was similar to that in wild-type mice at room temperature. However, at thermoneutrality, in which the regulation of heart rate switches from sympathetic to parasympathetic in wild-type mice, TRalpha1+m mice maintained sympathetic stimulation and failed to activate parasympathetic signaling. Our findings demonstrate a novel role for TRalpha1 in the adaptation of cardiac activity by the autonomic nervous system and suggest that human patients with a similar mutation in TRalpha1 might exhibit a deficit in cardiac adaptation to stress or physical activity and an increased sensitivity to beta-blockers.

Hypermetabolism in mice caused by the central action of an unliganded thyroid hormone receptor alpha1.

Thyroid hormone, via its nuclear receptors TRalpha and TRbeta, controls metabolism by acting locally in peripheral tissues and centrally by regulating sympathetic signaling. We have defined aporeceptor regulation of metabolism by using mice heterozygous for a mutant TRalpha1 with low affinity to T3. The animals were hypermetabolic, showing strongly reduced fat depots, hyperphagia and resistance to diet-induced obesity accompanied by induction of genes involved in glucose handling and fatty acid metabolism in liver and adipose tissues. Increased lipid mobilization and beta-oxidation occurred in adipose tissues, whereas blockade of sympathetic signaling to brown adipose tissue normalized the metabolic phenotype despite a continued perturbed hormone signaling in this cell type. The results define a novel and important role for the TRalpha1 aporeceptor in governing metabolic homeostasis. Furthermore, the data demonstrate that a nuclear hormone receptor affecting sympathetic signaling can override its autonomous effects in peripheral tissues.

Blebbistatin specifically inhibits actin-myosin interaction in mouse cardiac muscle.

Blebbistatin is a powerful inhibitor of actin-myosin interaction in isolated contractile proteins. To examine whether blebbistatin acts in a similar manner in the organized contractile system of striated muscle, the effects of blebbistatin on contraction of cardiac tissue from mouse were studied. The contraction of paced intact papillary muscle preparations and shortening of isolated cardiomyocytes were inhibited by blebbistatin with inhibitory constants in the micromolar range (1.3-2.8 muM). The inhibition constants are similar to those previously reported for isolated cardiac myosin subfragments showing that blebbistatin action is similar in filamentous myosin of the cardiac contractile apparatus and isolated proteins. The inhibition was not associated with alterations in action potential duration or decreased influx through L-type Ca(2+) channels. Experiments on permeabilized cardiac muscle preparations showed that the inhibition was not due to alterations in Ca(2+) sensitivity of the contractile filaments. The maximal shortening velocity was not affected by 1 muM blebbistatin. In conclusion, we show that blebbistatin is an inhibitor of the actin-myosin interaction in the organized contractile system of cardiac muscle and that its action is not due to effects on the Ca(2+) influx and activation systems.

Lung dysfunction causes systemic hypoxia in estrogen receptor beta knockout (ERbeta-/-) mice.

Estrogen receptor beta (ERbeta) is highly expressed in both type I and II pneumocytes as well as bronchiolar epithelial cells. ERalpha is not detectable in the adult lung. Lungs of adult female ERbeta knockout (ERbeta-/-) mice have already been reported to have fewer alveoli and reduced elastic recoil. In this article, we report that, by 5 months of age, there are large areas of unexpanded alveoli in lungs of both male and female ERbeta-/- mice. There is increased staining for collagen and, by EM, abnormal clusters of collagen fibers are seen in the alveolar septa of ERbeta-/- mice. Immunohistochemical analysis and Western blotting with lung membrane fractions of ERbeta-/- mice revealed down-regulation of caveolin-1, increased expression of membrane type-1 metalloproteinase, matrix metalloproteinase 2 (active form), and tissue inhibitors of metalloproteinases 2. Hypoxia, measured by immunohistochemical analysis for hypoxia-inducible factor 1alpha and chemical adducts (with Hypoxyprobe), was evident in the heart, ventral prostate, periovarian sac, kidney, liver, and brain of ERbeta-/- mice under resting conditions. Furthermore, both male and female adult ERbeta-/- mice were reluctant to run on a treadmill and tissue hypoxia became very pronounced after exercise. We conclude that ERbeta is necessary for the maintenance of the extracellular matrix composition in the lung and loss of ERbeta leads to abnormal lung structure and systemic hypoxia. Systemic hypoxia may be responsible for the reported left and right heart ventricular hypertrophy and systemic hypertension in ERbeta-/- mice.

Developmental regulation of nerve and receptor mediated contractions of mammalian urinary bladder smooth muscle.

The development of nerve-induced activation, receptor properties and cellular signalling was examined by comparing the urinary bladder of new-born (0-2 days) and adult mice. Tissue strips were isolated and the effects of different neuromuscular agents on force were investigated during electrical field stimulation. The nerve-induced contractions of the urinary bladders from new-born mice were less influenced by desensitisation with alpha, beta-methylene ATP and more sensitive to scopolamine compared with those of the adult bladder. There were no differences in alpha, beta-methylene ATP or ATP responsiveness between adult and new-born tissue, showing that the lower purinergic component of the nerve-induced responses in the new-born mice was due to properties of the transmitter release rather than to a change in receptor function. Dose-response curves for carbachol revealed a lower peak response in new-born bladders compared with adults. The phasic component of the cholinergic contractions was pronounced and initiated at low carbachol concentrations in the new-born tissue. The carbachol contractions of both new-born and adult urinary bladder tissue were inhibited by the Rho kinase inhibitor Y27632 and by the protein kinase G activator 8-Br-cGMP. However, the sustained phase of carbachol contraction was significantly less sensitive to Y27632 and 8-Br-cGMP in new-born tissue. These results suggest that the receptor mediated calcium sensitisation mechanism is less prominent in new-born compared with adult mice and that the contractions of new-born bladders are less influenced by the nitric oxide (NO)-cGMP inhibitory pathway.

Phospholipase C and cAMP-dependent positive inotropic effects of ATP in mouse cardiomyocytes via P2Y11-like receptors.

ATP is released as a cotransmitter together with catecholamines from sympathetic nerves. In the heart ATP has been shown to cause a pronounced positive inotropic effect and may also act in synergy with beta-adrenergic agonists to augment cardiomyocyte contractility. The aim of the present study was to investigate the inotropic effects mediated by purinergic P2 receptors using isolated mouse cardiomyocytes. Stable adenine nucleotide analogs were used and the agonist rank order for adenine nucleotide stimulation of the mouse cardiomyocytes was AR-C67085>ATPgammaS>2-MeSATP>>>2-MeSADP=0, that fits the agonist profile of the P2Y11 receptor. ATPgammaS induced a positive inotropic response in single mouse cardiomyocytes. The response was similar to that for the beta1 receptor agonist isoproterenol. The most potent response was obtained using AR-C67085, a P2Y11 receptor agonist. This agonist also potentiated contractions in isolated trabecular preparations. The adenylyl cyclase blocker (SQ22563) and phospholipase C (PLC) blocker (U73122) demonstrated that both pathways were required for the inotropic response of AR-C67085. A cAMP enzyme immunoassay confirmed that AR-C67085 increased cAMP in the cardiomyocytes. These findings are in agreement with the P2Y11 receptor, coupled both to activation of IP3 and cAMP, being a major receptor for ATP induced inotropy. Analyzing cardiomyocytes from desmin deficient mice, Des-/-, with a congenital cardiomyopathy, we found a lower sensitivity to AR-C67085, suggesting a down-regulation of P2Y11 receptor function in heart failure. The prominent action of the P2Y11 receptor in controling cardiomyocyte contractility and possible alterations in its function during cardiomyopathy may suggest this receptor as a potential therapeutic target. It is possible that agonists for the P2Y11 receptor could be used to improve cardiac output in patients with circulatory shock and that P2Y11 receptor antagonist could be beneficial in patients with congestive heart failure (CHF).

Actions downstream of cyclic GMP/protein kinase G can reverse protein kinase C-mediated phosphorylation of CPI-17 and Ca²âº sensitization in smooth muscle.

Ca(2+) sensitivity of smooth muscle contraction is modulated by several systems converging on myosin light chain phosphatase (MLCP). Rho-Rho kinase is considered to inhibit MLCP via phosphorylation, whereas protein kinase C (PKC) induced sensitization has been shown to be dependent on phosphorylation of the inhibitory protein CPI-17. We have explored the interaction of cGMP-dependent protein kinase (PKG) with Ca(2+) sensitization pathways using permeabilized mouse smooth muscle. Three conditions giving approximately 50% of maximal active force were compared in small intestinal preparations: 1). Ca(2+)-activated unsensitized muscle (pCa 5.9 with Rho kinase inhibitor Y27632); 2). Rho-Rho kinase-sensitized muscle (pCa 6.1 with guanosine 5'-3-O-(thio)triphosphate); and 3). PKC-sensitized muscle (pCa 6.0 with Y27632 and PKC activator phorbol 12,13-dibutyrate). 8-Br-cGMP relaxed the sensitized muscles but had marginal effects on unsensitized preparations, showing that PKG reverses both PKC and Rho-mediated Ca(2+) sensitization. CPI-17 was present in permeabilized intestinal tissue. In PKC-sensitized preparations, CPI-17 phosphorylation decreased in response to 8-Br-cGMP. The rate of PKC-mediated phosphorylation in the presence of the MLCP inhibitor microcystin-LR was not influenced by 8-Br-cGMP. PKC-induced Ca(2+) sensitization also was reversed in vascular smooth muscle tissues (portal vein and femoral artery). We conclude that actions downstream of cGMP/PKG can reverse PKC-mediated phosphorylation of CPI-17 and Ca(2+) sensitization in smooth muscle.

Nerve induced responses and force-velocity relations of regenerated detrusor muscle after subtotal cystectomy in the rat.

AIMS: To study the pharmacological and mechanical properties of newly developed detrusor muscle after subtotal cystectomy, to explore if the regenerated detrusor has characteristics similar to the normal bladder base, from which it regenerated, or to the normal bladder body which it replaces. METHODS: Partial cystectomy was performed in female rats. Fifteen weeks later, detrusor strips were cut from supratrigonal and equatorial segments. Unoperated rats served as controls. Responses to field stimulation were obtained in the absence and presence of scopolamine, prazosin, and P2X1 blockade. Dose-response curves were obtained for carbachol, alpha,beta-methylene-ATP, and phenylephrine. Force-velocity data were obtained on maximally activated chemically skinned preparations. RESULTS: Maximal contractile response to field stimulation was 60% of that to high-K+ with no difference between strips from control and cystectomy bladders. Prazosin had no effect. Scopolamine decreased maximal response of supratrigonal strips to 62 +/- 6 (controls) and 61 +/- 4% (operated) of that without blocker. For equatorial strips the decrease was to 81 +/- 5 (controls) and 58 +/- 8% (operated). Frequency-response relations were obtained during blockade with scopolamine, alpha,beta-methylene-ATP, and prazosin. Supratrigonal strips showed a pronounced additional inhibition up to 40 Hz. Equatorial strips from controls were completely inhibited at all frequencies. Equatorial strips from operated bladders were inhibited up to 20 Hz but not at 40 and 60 Hz. Carbachol EC(50) values were similar in all groups. Maximum response to phenylephrine was 10-20% of high-K+ response. Maximal shortening velocity (Vmax) was similar in control supratrigonal and equatorial strips, but was significantly lower in the operated bladders. CONCLUSIONS: (1): A regional difference exists in pharmacological properties of control detrusor, with a considerable contractile response to stimulation remaining in the supratrigonal muscle after simultaneous cholinergic, adrenergic, and purinergic blockade. (2): The new detrusor was functionally well innervated with no supersensitivity to muscarinic stimulation. (3): The newly formed bladder body had pharmacological properties specific for the supratrigonal segment from which it had developed. (4): There was no regional difference in force-velocity characteristics of the control detrusor. (5): The lowered Vmax in the newly formed bladder might thus be related to growth and regeneration of muscle cells.