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The cardiovascular complications following pulmonary exposure to a variety of unrelated materials can manifest as adverse outcome events. While the specific underlying mechanisms are yet to be fully elucidated the cardiovascular system response to exposures is manifested as a more robust response. In this chapter we describe cardiovascular responses to ambient particulate matter (PM) CeO2, Printex 90 (P90), and multi-walled carbon nanotube (MWCNT) nanoparticles. The inherent complexity of ambient sources of PM and the growing diversity of specifically engineered PM reinforce a need to understand the broader impact of exposures. Mice were exposed to 100 μg of PM or vehicle by intratracheal instillation. MWCNT, P90, and ambient PM all increased the ischemic-reperfusion injury compared to vehicle. Ambient PM and P90 decreased total blood leukocyte counts, while MWCNT induced a larger increase in plasma fibrinogen than ambient PM. Only ambient PM affected blood palette numbers and endothelial mediated relaxations. MWCNT exposure augmented the constrictor response but had no effect on endothelial dependent responses. These results illustrate our increasing yet limited understanding of the integrative nature of the cardiovascular system with neural, humeral, and immune systems and only begins to hint at the importance of a hypersensitive state on physiological and pathophysiological processes.

Background Although granulomatous inflammation is a central feature of many disease processes, cellular mechanisms of granuloma formation and persistence are poorly understood. Carbon nanoparticles, which can be products of manufacture or the environment, have been associated with granulomatous disease. This paper utilizes a previously described carbon nanoparticle granuloma model to address the issue of whether peroxisome proliferator-activated receptor gamma (PPARγ), a nuclear transcription factor and negative regulator of inflammatory cytokines might play a role in granulomatous lung disease. PPARγ is constitutively expressed in alveolar macrophages from healthy individuals but is depressed in alveolar macrophages of patients with sarcoidosis, a prototypical granulomatous disease. Our previous study of macrophage-specific PPARγ KO mice had revealed an intrinsically inflammatory pulmonary environment with an elevated pro-inflammatory cytokines profile as compared to wild-type mice. Based on such observations we hypothesized that PPARγ expression would be repressed in alveolar macrophages from animals bearing granulomas induced by MWCNT instillation. Methods Wild-type C57Bl/6 and macrophage-specific PPARγ KO mice received oropharyngeal instillations of multiwall carbon nanotubes (MWCNT) (100 μg). Bronchoalveolar lavage (BAL) cells, BAL fluids, and lung tissues were obtained 60 days post-instillation for analysis of granuloma histology and pro-inflammatory cytokines (osteopontin, CCL2, and interferon gamma [IFN-γ] mRNA and protein expression. Results In wild-type mice, alveolar macrophage PPARγ expression and activity were significantly reduced in granuloma-bearing animals 60 days after MWCNT instillation. In macrophage-specific PPARγ KO mice, granuloma formation was more extensive than in wild-type at 60 days after MWCNT instillation. PPARγ KO mice also demonstrated elevated pro-inflammatory cytokine expression in lung tissue, laser-microdissected lung granulomas, and BAL cells/fluids, at 60 days post MWCNT exposure. Conclusions Overall, data indicate that PPARγ deficiency promotes inflammation and granuloma formation, suggesting that PPARγ functions as a negative regulator of chronic granulomatous inflammation.

Background The exceptional physical-chemical properties of carbon nanotubes have lead to their use in diverse commercial and biomedical applications. However, their utilization has raised concerns about human exposure that may predispose individuals to adverse health risks. The present study investigated the susceptibility to cardiac ischemic injury following a single exposure to various forms of multi-walled carbon nanotubes (MWCNTs). It was hypothesized that oropharyngeal aspiration of MWCNTs exacerbates myocardial ischemia and reperfusion injury (I/R injury). Methods Oropharyngeal aspiration was performed on male C57BL/6J mice with a single amount of MWCNT (0.01 - 100 μg) suspended in 100 μL of a surfactant saline (SS) solution. Three forms of MWCNTs were used in this study: unmodified, commercial grade (C-grade), and functionalized forms that were modified either by acid treatment (carboxylated, COOH) or nitrogenation (N-doped) and a SS vehicle. The pulmonary inflammation, serum cytokine profile and cardiac ischemic/reperfusion (I/R) injury were assessed at 1, 7 and 28 days post-aspiration. Results Pulmonary response to MWCNT oropharyngeal aspiration assessed by bronchoalveolar lavage fluid (BALF) revealed modest increases in protein and inflammatory cell recruitment. Lung histology showed modest tissue inflammation as compared to the SS group. Serum levels of eotaxin were significantly elevated in the carboxylated MWCNT aspirated mice 1 day post exposure. Oropharyngeal aspiration of all three forms of MWCNTs resulted in a time and/or dose-dependent exacerbation of myocardial infarction. The severity of myocardial injury varied with the form of MWCNTs used. The N-doped MWCNT produced the greatest expansion of the infarct at any time point and required a log concentration lower to establish a no effect level. The expansion of the I/R injury remained significantly elevated at 28 days following aspiration of the COOH and N-doped forms, but not the C-grade as compared to SS. Conclusion Our results suggest that oropharyngeal aspiration of MWCNT promotes increased susceptibility of cardiac tissue to ischemia/reperfusion injury without a significant pulmonary inflammatory response. The cardiac injury effects were observed at low concentrations of MWCNTs and presence of MWCNTs may pose a significant risk to the cardiovascular system.

The use and production of multi-walled carbon nanotubes (MWCNTs) have significantly increased over the last decade due to their versatility in numerous applications. Their unique physical and chemical properties make them desirable for various biomedical applications, but the same properties also raise concerns about their safety to human health, particularly at the cellular level. The vascular endothelium could be exposed to nanomaterials either by direct intravenous administration in nanomedicine or by translocation following inhalational exposure in an occupational setting. We hypothesized that direct exposure to MWCNTs will increase the expression of inflammatory markers in human aortic endothelial cells (HAEC). We also investigated the effect of the route of exposure on activation by changing the suspension medium of the MWCNTs. HAEC were treated in vitro with MWCNTs (1 or 10 μg/cm2) suspended in either cell culture medium [(M)-MWCNTs] or 10% clinical grade pulmonary surfactant [(S)-MWCNTs]. The zeta potential of the (S)-MWCNTs was significantly more negative than the (M)-MWCNTs suggesting a more stable suspension. Treatment of HAEC with (S)-MWCNTs; as compared to (M)-MWCNTs resulted in a significantly higher up-regulation of mRNA transcripts for cell adhesion molecules VCAM1, SELE, ICAM1 and the chemokine CCL2. Time dependent changes in VCAM1 and CCL2 protein levels were confirmed by immunofluorescence, flow cytometry and ELISA. A label free quantitative mass spectrometry proteomic analysis was utilized to compare protein expression patterns between the two suspensions of MWCNTs. We identified significant expression changes in >200 unique proteins in MWCNT treated HAEC. However, the two suspensions of MWCNTs resulted in different protein expression patterns with the eIF2 pathway as the only common pathway identified between the two suspensions. These data suggest that direct exposure to MWCNTs induces acute inflammatory and protein expression changes in HAEC, which is influenced by the type of media used for suspension of MWCNTs and their resulting zeta potential.

1. Ca(2+)-dependent phosphorylation of the myosin regulatory light chain (MRLC) initiates cross-bridge cycling and contraction in smooth muscle. A four-state cross-bridge model, in which Ca(2+)-dependent phosphorylation is the only proposed regulatory mechanism, can predict the mechanical output of the swine carotid media. Our aims were to determine whether ATP consumption rates and the economy of force maintenance are regulated functions of MRLC phosphorylation as predicted by the model. 2. Steady-state force and oxygen consumption were measured in medial rings of swine carotid arteries activated with depolarizing solutions and agents capable of maintaining a wide range of steady-state myoplasmic Ca2+ and MRLC phosphorylation levels. 3. Suprabasal ATP consumption increased almost linearly with MRLC phosphorylation and exhibited a hyperbolic increase with active stress, as predicted. 4. The economy of stress maintenance fell with increases in suprabasal phosphorylation. 5. In absolute terms the energetic cost of covalent regulation by cross-bridge phosphorylation was small, although it may be a significant fraction of the ATP consumption associated with contraction.

Recent observations have demonstrated that nanomaterials may be toxic to human tissue. While the ability of nano-scaled particulate matter is known to cause a range of problems in respiratory system, recent observations suggest that the nervous system may be vulnerable as well. In the current paper we asked whether exposure of primary neuronal cell cultures to nanoparticles might compromise regenerative axon growth. Regenerative response was triggered by performing a conditioning lesion of sciatic nerve five days prior to collection of dorsal root ganglia (DRG). DRG neurons were plated at a low density and incubated with multi-walled carbon nanotubes (MWCNT) (0.1 – 10 μg/ml in 10% of surfactant in saline) overnight. The experiments showed that exposure of DRG cultures to MWCNT significantly impaired regenerative axonogenesis without concomitant cell death. These results indicate that MWNCTs may have detrimental effect on nerve regeneration and may potentially trigger axonal pathology.

Asthma is often associated with cardiovascular complications, and recent observations in animal models indicate that induction of pulmonary allergic inflammation increases susceptibility of the myocardium to ischemia and reperfusion injury. In this study, we used a murine model of allergen sensitization in which aspiration of allergen induces pulmonary and systemic inflammation, to test the hypothesis that pulmonary exposure to allergen alters vascular relaxation responses. BALB/C mice were sensitized by intra-peritoneal injection of ragweed and challenged by intratracheal instillation of allergen. Airway hyperreactivity and pulmonary inflammation were confirmed, and endothelium-dependent and - independent reactivity of thoracic aorta rings were evaluated. Ragweed-sensitization and challenge induced airway hyperreactivity to methacholine and pulmonary inflammation, but did not affect constrictor responses of the aortic rings to phenylephrine and K depolarization. In contrast, maximal relaxation of aortic rings to acetylcholine and sodium nitroprusside decreased from 87.6 ± 3.9 % and 97.7 ± 1.2 % to 32 ± 4 % and 51 ± 6 %, respectively (p< 0.05). The sensitivity to acetylcholine was likewise reduced (EC50 = 0.26 ± 0.05 µM vs. 1.09 ± 0.16 µM, p < 0.001). The results demonstrate that induction of allergic pulmonary inflammation in mice depresses endothelium-dependent and -independent vascular relaxation, which can contribute to cardiovascular complications associated with allergic inflammation.

Congestive heart failure (CHF) alters vascular reactivity and an up regulation in Urotensin II (UTII), a potent vasoactive peptide. The aim of this study was to investigate the interaction between CHF and UTII in altering vascular reactivity in a rat model of volume overload heart failure. Animals were divided into 4 groups: control, UTII infused (UTII), volume overload only (VO) or volume overload+UTII (VO+UTII). Volume overload was established by the formation of an aorto-caval fistula. Following fistula formation animals were administered UTII at a rate of 300 pmol/kg/hr for 4 weeks subcutaneously with mini-osmotic pumps. Thoracic aorta rings, with/without endothelium, were subjected to cumulative dose-responses to phenylephrine, sodium nitroprusside (SNP), acetylcholine (ACH), UTII, and the Rho-kinase inhibitor HA-1077. Aortas from VO animals exhibited increased sensitivity to phenylephrine and UTII with a decreased relaxation response to ACH and HA-1077. Aortas from animals subjected to chronic UTII with volume overload (VO + UTII) retained their sensitivity to phenylephrine and UTII while they improved their relaxation to HA-1077 but not ACH. The constrictive response to UTII was dose-dependent and augmented at concentrations < 0.01 μM in VO animals. The changes in vascular reactivity paralleled an elevation of both the UTII and α1a-adrenergic receptor while the Rho and Rho-kinase signalling proteins were diminished. We found that volume overload increased sensitivity to the vasoconstrictor agents that was inversely related to changes in the Rho-kinase expression. The addition of UTII with VO reversed the constrictive vascular response through alterations in the Rho-kinase signalling pathway.

Increased ambient air particulate matter (PM) concentrations are associated with risk for myocardial infarction, stroke, and arrhythmia, and ultrafine PM (UFPM) might be particularly toxic to the cardiovascular system. Recent epidemiological studies are beginning to offer mechanistic insights, yet the rodent model remains a valuable tool to explore potential mechanisms. This article reviews a series of studies from our laboratory demonstrating the promise of mouse models to link health effects to biological mechanisms. Specifically, data from 6- to 10-wk-old male ICR mice exposed to intratracheal instillation of 100 microg of UFPM collected from the Chapel Hill, NC airshed are described. Studies of ischemia/reperfusion, vascular function, and hemostasis are described. In summary, UFPM exposure doubles the size of myocardial infarction attendant to an episode of ischemia and reperfusion while increasing postischemic oxidant stress. UFPM alters endothelial-dependent and -independent regulation of systemic vascular tone; increases platelet number, plasma fibrinogen, and soluble P-selectin levels; and reduces bleeding time, implying enhanced thrombogenic potential. Taking these findings together, this model of acute UFPM exposure in the mouse indicates that UFPM induces a prothrombotic state and decreases vasomotor responsiveness, thereby offering insight into how UFPM could contribute to vascular events associated with thrombosis and ischemia and increasing the extent of infarction.

Introduction The combination of the independent risk factors for erectile dysfunction, obesity, hypertension, and diabetes are manifested collectively in a condition known as metabolic syndrome X. However, the exact mechanism(s) by which the combination of these factors contributes to erectile dysfunction have yet to be elucidated. Aim We hypothesized that protein kinase C (PKC) and Rho-kinase enhanced vascular tone and thus contributed to erectile dysfunction in this condition. Methods Erectile function was evaluated by recording voltage-dependent increases in intracavernosal pressure following stimulation of the cavernosal nerve in 16- to 20-week-old lean and obese-diabetic Zucker rats. Cavernosal tissue contractile and relaxation responses were evaluated in vitro when contracted with phenylephrine, endothelin-1 and relaxed by Rho-kinase, PKC inhibitors or sodium nitroprusside. Additionally, cavernosal tissue Rho-kinase, protein kinase, and nitric oxide synthase isoform expression were evaluated by Western blot. Results The voltage-dependent erectile responses were suppressed by >30% in the obese-diabetic Zucker rat. The maximal stress generated by cavernosal tissue from the obese-diabetic was significantly greater than the lean response by greater than 0.8 mN/mm 2 for both phenylephrine and endothelin stimulation. The PKC inhibitor, chelerythrine, inhibited more than 30% of the phenylephrine-induced and 70% of the endothelin-1-induced contractions. Rho-kinase inhibition, with either Y-27632 or HA-1077, revealed impaired relaxations of nearly 30% in tissue from obese-diabetic animals. Western blot analysis revealed increased protein expression of PKC α and δ and Rho-kinase α and β but no loss for endothelial or neuronal nitric oxide synthase. Conclusions In this rodent model both PKC and Rho-kinase signaling elements may contribute to an enhanced vasoconstriction state of the penile smooth muscle that was differentially dependent on the agonist used. The enhanced vasoconstrictive state of the tissue could contribute to the reduced voltage-dependent erectile response in the obese-diabetic Zucker rat. Wingard C, Fulton D, and Husain S. Altered penile vascular reactivity and erection in the Zucker obese-diabetic rat.

Epidemiological studies have linked levels of particulate matter (PM) in ambient air to cardiovascular mortality and hospitalizations for myocardial infarction (MI) and stroke. Thrombus formation plays a primary role in potentiating acute cardiovascular events, and this study was undertaken to determine whether pulmonary exposure to PM alters hemostasis. PM was collected from the Chapel Hill, NC airshed and was administered to mice by intratracheal instillation at a dose previously shown to exacerbate myocardial ischemia-reperfusion injury. Twenty-four hours after exposure, an increase occurred in the number of circulating platelets and plasma concentrations of fibrinogen and soluble P-selectin. The concentration of tissue factor pathway inhibitor (TFPI) in plasma was decreased, whereas the plasma concentration of plasminogen activator inhibitor (PAI-1) was increased. Consistent with these observations, bleeding time from a tail-tip transection was shortened. These results provide evidence that PM exposure alters hemostasis in otherwise healthy animals and may thereby promote clot formation and impede clot resolution in susceptible individuals. The results also establish definite hemostatic endpoints that can be used to further investigate the effects of dose and particle characteristics on the toxicity of ambient particles.

Epidemiological studies have linked ambient particulate matter (PM) levels to an increased incidence of adverse cardiovascular events. Yet little is definitively known about the mechanisms accounti...

Serine 19 phosphorylation of the myosin regulatory light chain (MRLC) appears to be the primary determinant of smooth muscle force development. The relationship between MRLC phosphorylation and force is nonlinear, showing that phosphorylation is not a simple switch regulating the number of cycling cross bridges. We reexamined the MRLC phosphorylation-force relationship in slow, tonic swine carotid media; fast, phasic rabbit urinary bladder detrusor; and very fast, tonic rat anococcygeus. We found a sigmoidal dependence of force on MRLC phosphorylation in all three tissues with a threshold for force development of ∼0.15 mol Pi/mol MRLC. This behavior suggests that force is regulated in a highly cooperative manner. We then determined whether a model that employs both the latch-bridge hypothesis and cooperative activation could reproduce the relationship between Ser19-MRLC phosphorylation and force without the need for a second regulatory system. We based this model on skeletal muscle in which attached cross bridges cooperatively activate thin filaments to facilitate cross-bridge attachment. We found that such a model describes both the steady-state and time-course relationship between Ser19-MRLC phosphorylation and force. The model required both cooperative activation and latch-bridge formation to predict force. The best fit of the model occurred when binding of a cross bridge cooperatively activated seven myosin binding sites on the thin filament. This result suggests cooperative mechanisms analogous to skeletal muscle that will require testing.

We have observed a lengthening of the duration between spontaneous cardiac contractions under conditions that preferentially activate the epsilon protein kinase C (epsilonPKC) isozyme. Therefore, we investigated whether this response could be selectively mediated by epsilonPKC in neonatal cardiac myocytes (NCMs) and adult rat ventricular trabeculae. Contraction of NCMs was monitored using light scattering techniques and trabecular force generation was monitored in tissue baths using a force transducer. The involvement of the epsilonPKC isozyme was confirmed using an epsilonPKC-selective translocation inhibitor and Western blot translocation assays. In NCMs 3 nM 4-beta phorbol 12-myristate-13-acetate (PMA) treatment preferentially activates (translocates) epsilonPKC. In this study 3 nM 4-beta PMA induced a 2-fold increase in contractile amplitude and a approximately 14-fold increase in the quiescent period between contractions in NCMs. Extracellular adenosine 5'-triphosphate (ATP) also enhanced contractile amplitude by 1.7-fold and the quiescent period duration by 8-fold. The enhancement of quiescent period duration was attenuated by an epsilonPKC-selective translocation inhibitor. To investigate these relationships in intact myocardium, we studied spontaneously beating adult rat ventricular trabecula. In these fibers contractile amplitude was only modestly enhanced; however, the quiescent period was lengthened by 4.5-fold following a 15-min exposure to 3 nM 4-beta PMA. 4-beta PMA treatment also promoted arrhythmogenesis and increased the association of epsilonPKC with the particulate fraction in these fibers. Our results suggest that epsilonPKC may influence a specific phase of ventricular myocyte spontaneous beating. A better understanding of epsilonPKC modulation of spontaneous cardiac contraction may improve our understanding of the molecular events contributing to ventricular automaticity.

Recent studies have suggested that contraction of the smooth muscle in the cavernosal arterioles and in the walls of the cavernosal sinuses is maintained by the RhoA/Rho-kinase signaling pathway. However, this contraction activity must be overcome to permit the vasorelaxation essential for erection. We postulate that nitric oxide (NO) causes erection primarily by inhibiting the RhoA/Rho-kinase pathway. The following will discuss evidence in support of the important role of Rho-kinase-mediated vasoconstriction in the nonerect penis and how NO overrides this Rho-kinase-mediated vasoconstriction to permit vasodilation and erection.

Maintenance of the detumescent state of the penis is believed to involve the actions of several vasoconstrictors. However, our mechanistic understanding of any synergistic vasoconstrictor influences is extremely limited. We tested the hypothesis that a vasoconstrictor combination of endothelin (ET-1) and phenylephrine (PE) augments the constrictor responses in rat corporal cavernosal tissues by a mechanism involving the RhoA-Rho kinase pathway. Independently, ET-1 (1 nM-30 μM) and PE (100 nM-100 μM) both caused dose-dependent contractions of isolated rat cavernosal tissues. In combination, ET-1 (30 nM) augmented the contractile effect of PE and shifted the calculated EC50 for PE (90 ± 12 to 45 ± 5 μM). The active stress generated by cavernosal strips during the ET-1 + PE combined stimulation (4.9 ± 0.2 mN/mm2) was greater than the combined stress generated with ET-1 (0.4 ± 0.1 mN/mm2) or PE (3.3 ± 0.2 mN/mm2) stimulations alone. Blockade of ETA receptors (30 nM; A-127722) reversed the augmented stress generation and the Rho-kinase inhibitor Y-27632 differentially and dose-dependently relaxed the tissue. The combined constrictor effect was associated with a fourfold increase of RhoA in the membrane faction of the tissue homogenates. We conclude that the ET-1 + PE combination potentiate vasoconstriction through mutual activation of the RhoA-Rho kinase pathway. The interactions of these agonists likely play important roles in the maintenance of the flaccid state and contribute to some forms of erectile dysfunction.

Ablation of the premigratory cardiac neural crest (CNC) from the chick embryo results in a malformed outflow tract vasculature termed persistent truncus arteriosus (PTA). In addition, loss of the CNC disrupts myocardial excitation-contraction (EC) coupling, decreases intracellular Ca2+ transients, and depresses force generation. We examined if similar defects occurred in the neural crest-derived smooth muscle of the aortic arch in a test of the hypothesis that loss of elements from the CNC disrupts EC coupling and force production in the smooth muscle of the tunica media of the aortic arch. Aortic arch segments from chicks (embryonic day 15) displaying PTA generated approximately 43% of stress generated by the aortic arch from sham-operated control embryos during potassium depolarization. The depressed force response was associated with a twofold lower Fura-2 transient. In contrast, force and steady-state Fura-2 signals during endothelin-1 stimulation were unchanged. The differences seen in stress generation with potassium depolarization between sham and PTA displaying embryos were not seen in the descending aorta, a tissue not derived from the neural crest. Protein content and immunostaining revealed no differences in the content of actin, myosin, or dihydropyridine receptor from sham or PTA aortic arch. Our results suggest that the CNC is required for normal aortic arch smooth muscle function and support the hypothesis that the loss of CNC impacts the force generating ability, in part by disruption of the EC-coupling processes and altering Ca(2+)-handling.

Phosphatidylinositol 3-kinase (PI3-kinase) activates protein kinase B (also known as Akt), which phosphorylates and activates a cyclic nucleotide phosphodiesterase 3B. Increases in cyclic nucleotide concentrations inhibit agonist-induced contraction of vascular smooth muscle. Thus we hypothesized that the PI3-kinase/Akt pathway may regulate vascular smooth muscle tone. In unstimulated, intact bovine carotid artery smooth muscle, the basal phosphorylation of Akt was higher than that in cultured smooth muscle cells. The phosphorylation of Akt decreases in a time-dependent manner when incubated with the PI3-kinase inhibitor, LY-294002. Agonist (serotonin)-, phorbol ester (phorbol 12,13-dibutyrate; PDBu)-, and depolarization (KCl)-induced contractions of vascular smooth muscles were all inhibited in a dose-dependent fashion by LY-294002. However, LY-294002 did not inhibit serotonin- or PDBu-induced increases in myosin light chain phosphorylation or total O2consumption, suggesting that inhibition of contraction was not mediated by reversal or inhibition of the pathways that lead to smooth muscle activation and contraction. Treatment of vascular smooth muscle with LY-294002 increased the activity of cAMP-dependent protein kinase and increased the phosphorylation of the cAMP-dependent protein kinase substrate heat shock protein 20 (HSP20). These data suggest that activation of the PI3-kinase/Akt pathway in unstimulated smooth muscle may modulate vascular smooth muscle tone (allow agonist-induced contraction) through inhibition of the cyclic nucleotide/HSP20 pathway and suggest that cyclic nucleotide-dependent inhibition of contraction is dissociated from the myosin light chain contractile regulatory pathways.

Recent evidence indicates that endothelin-1 (ET-1) might be a principal vasoconstrictor in the penis. We report that ET-1 injection into the cavernous sinuses before erection sharply reduced the magnitude of subsequent erections. Corpus cavernosum pressure-to-mean arterial pressure ratios (CCP/MAP), with maximal ganglionic stimulation, were 0.62 ± 0.05 before ET-1 injection and 0.31 ± 0.05 after, indicating that ET-1 acted as a vasoconstrictor. When ET-1 was injected during a maximal neurally induced erection, the ability of ET-1 to attenuate subsequent erections was diminished (CCP/MAP 0.75 ± 0.02 before ET-1, 0.61 ± 0.03 after). At submaximal stimulation voltages, injection of ET-1 during erection also attenuated its vasoconstrictive effect. Similarly, when ET-1 was injected during erection induced by intracavernosal injection of the nitric oxide (NO) donor NOR-1, subsequent erections were not significantly suppressed (CCP/MAP 0.53 ± 0.04 before ET-1, 0.45 ± 0.04 after). These findings that ET-1-induced vasoconstriction is attenuated during erection are consistent with the hypothesis that NO mediates erection both by initiating pathways that cause smooth muscle relaxation and by inhibiting the vasoconstrictive actions of ET-1.

Relaxation of the smooth muscle cells in the cavernosal arterioles and sinuses results in increased blood flow into the penis, raising corpus cavernosum pressure to culminate in penile erection. Nitric oxide, released from non-adrenergic/non-cholinergic nerves, is considered the principle stimulator of cavernosal smooth muscle relaxation, however, the inhibition of vasoconstrictors (that is, norepinephrine and endothelin-1, refs. 5-9) cannot be ignored as a potential regulator of penile erection. The calcium-sensitizing rho-A/Rho-kinase pathway may play a synergistic role in cavernosal vasoconstriction to maintain penile flaccidity. Rho-kinase is known to inhibit myosin light chain phosphatase, and to directly phosphorylate myosin light-chain (in solution), altogether resulting in a net increase in activated myosin and the promotion of cellular contraction. Although Rho-kinase protein and mRNA have been detected in cavernosal tissue, the role of Rho-kinase in the regulation of cavernosal tone is unknown. Using pharmacologic antagonism (Y-27632, ref. 13, 18), we examined the role of Rho-kinase in cavernosal tone, based on the hypothesis that antagonism of Rho-kinase results in increased corpus cavernosum pressure, initiating the erectile response independently of nitric oxide. Our finding, that Rho-kinase antagonism stimulates rat penile erection independently of nitric oxide, introduces a potential alternate avenue for the treatment of erectile dysfunction.

Studies in this laboratory are designed to determine the effects of vasoconstrictor agents on the erectile response in rats. We have previously demonstrated that the vasoconstrictor effect of endothelin-1 (ET-1) is sharply reduced by erection and by nitric oxide (NO) administration. The present study was performed to determine if vasoconstriction, resulting from alpha-adrenergic stimulation, is altered by erection and NO. During continuous monitoring of corpus cavernosum pressure (CCP) and mean arterial pressure (MAP), erection was induced by electrical stimulation of the autonomic ganglion for the innervation of the penis. When the alpha-adrenergic agonist methoxamine (METH, 10 microg/kg) was injected before erection (ie, into the non-erect penis), the subsequent erectile response (CCP/MAP) was significantly reduced from 0.68+/-0.03 before METH to 0.34+/-0.08 after METH. Injection of METH into the erect penis (ie, during erection) reduced the vasoconstrictor action of METH; CCP/MAP was 0.74+/-0.02 before METH and 0.55+/-0.05 after METH (P

Specific receptor antagonists were used to examine the role of endothelin-1 (ET-1) in the erectile response of the rat. In these studies, intact rats were cannulated to permit the continuous recording of mean arterial pressure (MAP) and intracavernosal pressure (CCP). Erection was induced by electrical stimulation of the autonomic ganglion, which regulates blood flow to the penis. The animals were subjected to intracavernosal injection with vehicle only (Cont) or with an antagonist to the endothelin-A receptor (ETA) or to the endothelin-B receptor (ETB). Blockade of the ETAor the ETBhad no effect on the erectile response (CCP/MAP) during maximal ganglionic stimulation. When ET-1 was injected into Cont rats, there was a marked vasoconstriction with a sharp rise in MAP and a decline in CCP as the cavernosal arterioles constricted and limited inflow. The injection of the ETAantagonist prevented the vasoconstriction after ET-1 injection into Cont rats, whereas blockade of the ETBhad no effect on the vasoconstrictive effect to ET-1. Similar results were obtained during submaximal ganglionic stimulation. With minimal levels of ganglionic stimulation, ET-1 injection led to a moderated degree of vasodilation in the presence of the ETAantagonist. The ETBantagonist failed to alter the CCP response during minimal stimulation, but it did have a marked effect on the MAP response to ET-1 injection. The results of these studies confirm that cavernosal tissue of the rat penis is highly responsive to ET-1. However, the failure of the ET-1 antagonists to affect penile erection in response to ganglionic stimulation reflects a minimal role of ET-1 in the erectile response in the rat.

Nitrovasodilators such as nitroglycerine, via production of nitric oxide and an increase in [cGMP], can induce arterial smooth muscle relaxation without proportional reduction in myosin light chain (MLC) phosphorylation or myoplasmic [Ca2+]. These findings suggest that regulatory systems, other than MLC phosphorylation and Ca2+, partially mediate nitroglycerine-induced relaxation. In swine carotid artery, we found that a membrane-permeant cGMP analogue induced relaxation without MLC dephosphorylation, suggesting that cGMP mediated the relaxation. Nitroglycerine-induced relaxation was associated with a reduction in O2 consumption, suggesting that the interaction between phosphorylated myosin and the thin filament was inhibited. Nitroglycerine-induced relaxation was associated with a 10-fold increase in the phosphorylation of a protein on Ser16. We identified this protein as heat shock protein 20 (HSP20), a member of a family of proteins known to bind to thin filaments. When homogenates of nitroglycerine-relaxed tissues were centrifuged at 6000 g, phosphorylated HSP20 preferentially sedimented in the pellet, suggesting that phosphorylation of HSP20 may increase its affinity for the thin filament. We noted that a domain of HSP20 is partially homologous to the 'minimum inhibitory sequence' of skeletal troponin I. The peptide HSP20110-121, which contains this domain, bound to actin-containing filaments only in the presence of tropomyosin, a characteristic of troponin I. High concentrations of HSP20110-121 abolished Ca2+-activated force in skinned swine carotid artery. HSP20110-121 also partially decreased actin-activated myosin S1 ATPase activity. These data suggest that cGMP-mediated phosphorylation of HSP20 on Ser16 may have a role in smooth muscle relaxation without MLC dephosphorylation. HSP20 contains an actin-binding sequence at amino acid residues 110-121 that inhibited force production in skinned carotid artery. We hypothesize that phosphorylation of HSP20 regulates force independent of MLC phosphorylation via binding of HSP20 to thin filaments and inhibition of cross-bridge cycling.

Experiments were designed to examine the efficacy of the MLCK inhibitors wortmannin and ML-9 in intact smooth muscle to determine whether contractile agonists can induce a Ca(2+) and myosin light chain phosphorylation-independent contraction. Both wortmannin and ML-9 reduced active stress in a dose-dependent manner. Both inhibitors interfered with Ca2+ mobilization in either the K(+)-depolarized or agonist activated swine carotid media at concentrations greater than 10 microM. Wortmannin reduced MRLC phosphorylation and stress to resting levels in stimulated tissues while Ca2+ remained above resting levels. There was no evidence for Ca2+ and MRLC phosphorylation-independent stress generation in swine arterial smooth muscle.

1. The objective of this study was to partition the increase in ATP consumption during contraction of swine carotid arterial smooth muscle estimated from suprabasal oxygen consumption (suprabasal JO2) and lactate release (Jlactate) into a component associated with cross-bridge cycling (JX) and one reflecting activation (JA). 2. Two experimental approaches-varying length under constant activation, and varying activation at a long length (1.8 times the optimal length for force development (Lo)) where force generation is minimal-revealed a linear dependence of JO2 and activation energy (JA) on cross-bridge phosphorylation. Protocols inducing a large increase in myosin regulatory light chain (MRLC) phosphorylation at 1.8 Lo resulted in significant elevations of JO2 and marked reductions in the economy of force maintenance. Our evidence suggests that this is primarily due to the increased cost of cross-bridge phosphorylation. 3. The extrapolated estimate of JA during maximal K(+)-induced depolarization made by varying length was 16%, while at 1.8 Lo it was 33% of the suprabasal JO2 at Lo. Calculated activation energies ranged from 17 to 45% of the suprabasal JO2 at Lo and from 72 to 87% of the suprabasal JO2 at 1.8 Lo under stimulation conditions that varied steady-state MRLC phosphorylation from 15 to 50%. 4. The results suggest that the kinetics of cross-bridge phosphorylation-dephosphorylation can rival those of cross-bridge cycling during isometric contractions in swine arterial smooth muscle.

1. The dependence of force (F) on length (L) in smooth muscle remains uncertain since (i) it is influenced by changes in activation (myosin light chain phosphorylation), (ii) no anatomical reference length for the contractile unit is available, (iii) the length at which optimum force is generated (L(o)) exhibits a broad, flat optimum, and (iv) the presence of an extensive connective tissue network makes it difficult to stretch tissues without damage. 2. A swine carotid medial ring preparation prepared by removal of the adventitia and endothelium could be stretched to 1.8 L(o) without decreasing active force generation on return to shorter lengths. 3. A highly reproducible mechanically defined reference length, L(o), was obtained by fitting force-length data between 0.3 and 1.6 L(o) with a third-order polynomial where L = L(o) when dF/dL = 0. 4. Activation as assessed by myosin regulatory light chain (MRLC) phosphorylation increased with length in 100 microM histamine-stimulated tissues from 0.6 to 1.8 L(o). 5. Activation was constant in K(+)-depolarized and field-stimulated tissues from 1.0 to 1.8 L(o) allowing determination of the descending limb of the force-length relation to be assessed independently of activation. 6. The slope of the descending limb of the force-length relation was linear except at very long lengths, which often produced tissue damage. The slope was not statistically different from that estimated for sarcomeres in vertebrate skeletal muscle. 7. The medial ring preparation and the procedures used to define the reference length provide advantages for the measurement of length-dependent variables.

Background Multi-walled carbon nanotubes (MWCNTs) are widely used in many disciplines due to their unique physical and chemical properties. Therefore, some concerns about the possible human health and environmental impacts of manufactured MWCNTs are rising. We hypothesized that instillation of MWCNTs impairs pulmonary function in C57BL/6 mice due to development of lung inflammation and fibrosis. Methods MWCNTs were administered to C57BL/6 mice by oropharyngeal aspiration (1, 2, and 4 mg/kg) and we assessed lung inflammation and fibrosis by inflammatory cell infiltration, collagen content, and histological assessment. Pulmonary function was assessed using a FlexiVent system and levels of Ccl3, Ccl11, Mmp13 and IL-33 were measured by RT-PCR and ELISA. Results Mice administered MWCNTs exhibited increased inflammatory cell infiltration, collagen deposition and granuloma formation in lung tissue, which correlated with impaired pulmonary function as assessed by increased resistance, tissue damping, and decreased lung compliance. Pulmonary exposure to MWCNTs induced an inflammatory signature marked by cytokine (IL-33), chemokine (Ccl3 and Ccl11), and protease production (Mmp13) that promoted the inflammatory and fibrotic changes observed within the lung. Conclusions These results further highlight the potential adverse health effects that may occur following MWCNT exposure and therefore we suggest these materials may pose a significant risk leading to impaired lung function following environmental and occupational exposures.

Background beta-defensins are small cationic peptides that exhibit broad spectrum antimicrobial properties. The majority of beta-defensins identified in humans are predominantly expressed in the male reproductive tract and have roles in non-immunological processes such as sperm maturation and capacitation. Characterization of novel defensins in the male reproductive tract can lead to increased understanding of their dual roles in immunity and sperm maturation. Methods In silico rat genomic analyses were used to identify novel beta-defensins related to human defensins 118–123. RNAs isolated from male reproductive tract tissues of rat were reverse transcribed and PCR amplified using gene specific primers for defensins. PCR products were sequenced to confirm their identity. RT-PCR analysis was performed to analyze the tissue distribution, developmental expression and androgen regulation of these defensins. Recombinant defensins were tested against E. coli in a colony forming unit assay to analyze their antimicrobial activities. Results Novel beta-defensins, Defb21, Defb24, Defb27, Defb30 and Defb36 were identified in the rat male reproductive tract. Defb30 and Defb36 were the most restricted in expression, whereas the others were expressed in a variety of tissues including the female reproductive tract. Early onset of defensin expression was observed in the epididymides of 10–60 day old rats. Defb21-Defb36 expression in castrated rats was down regulated and maintained at normal levels in testosterone supplemented animals. DEFB24 and DEFB30 proteins showed potent dose and time dependent antibacterial activity. Conclusion Rat Defb21, Defb24, Defb27, Defb30 and Defb36 are abundantly expressed in the male reproductive tract where they most likely protect against microbial invasion. They are developmentally regulated and androgen is required for full expression in the adult epididymis.

Chironomidae larvae were collected from the upper Tuscarawas River (Stark and Summit Counties, Ohio) in late spring and summer of 1985. Samples were taken from the benthos by shovel and from multiplate samplers at seven stations along a 13 Km portion of the river. Over 1, 100 larvae were identified. Fifty four taxa were recognized including 35 species, 7 species—groups, and 12 genera. Most taxa collected have a widespread distribution in North America. Two taxa, Procladius subletti and Diamesa, are generally reported from more northern regions and were unexpected in the Tuscarawas River. Additionally, the community diversity index reveals a large difference in community structure for those samples collected by shovel sampler compared to those collected by multiplate samplers.