Effects of carvedilol on human prostate tissue contractility and stromal cell growth pointing to potential clinical implications.

BACKGROUND: Apart from antagonizing ß-adrenoceptors, carvedilol antagonizes vascular α1-adrenoceptors and activates G protein-independent signaling. Even though it is a commonly used antihypertensive and α1-adrenoceptors are essential for the treatment of voiding symptoms in benign prostatic hyperplasia, its actions in the human prostate are still unknown. Here, we examined carvedilol effects on contractions of human prostate tissues, and on stromal cell growth. METHODS: Contractions of prostate tissues from radical prostatectomy were induced by electric field stimulation (EFS) or α1-agonists. Growth-related functions were examined in cultured stromal cells. RESULTS: Concentration-response curves for phenylephrine, methoxamine and noradrenaline were right shifted by carvedilol (0.1-10 µM), around half a magnitude with 100 nM, half to one magnitude with 1 µM, and two magnitudes with 10 µM. Right shifts were reflected by increased EC50 values for agonists, with unchanged Emax values. EFS-induced contractions were reduced by 21-54% with 0.01-1 µM carvedilol, and by 94% by 10 µM. Colony numbers of stromal cells were increased by 500 nM, but reduced by 1-10 µM carvedilol, while all concentrations reduced colony size. Decreases in viability were time-dependent with 0.1-0.3 µM, but complete with 10 µM. Proliferation was slightly increased by 0.1-0.5 µM, but reduced with 1-10 µM. CONCLUSIONS: Carvedilol antagonizes α1-adrenoceptors in the human prostate, starting with concentrations in ranges of known plasma levels. In vitro, effect sizes resemble those of α1-blockers used for the treatment of voiding symptoms, which requires concentrations beyond plasma levels. Bidirectional and dynamic effects on the growth of stromal cells may be attributed to "biased agonism".

Perinatal hypoxia weakens anticontractile influence of NO in rat arteries during early postnatal period.

BACKGROUND: Perinatal hypoxia affects a lot of neonates worldwide every year, however its effects on the functioning of systemic circulation are not clear yet. We aimed at investigation the effects of perinatal hypoxia on the second day of life on the functioning of the rat systemic vasculature in early postnatal period. METHODS: 2-day-old male rat pups were exposed to normobaric hypoxia (8% O2, 92% N2) for 2 hours. At the 11-14 days cutaneous (saphenous) arteries were isolated and studied by wire myography and Western blotting. RESULTS: Hypoxia weakened the contribution of anticontractile influence of NO, but did not affect the contribution of Rho-kinase or Kv7 channels to the contraction to α1-adrenergic agonist methoxamine. The content of eNOS and protein kinase G were not altered by hypoxic conditions. CONCLUSION: Perinatal hypoxia in rats at the second day of life leads to the decrease of anticontractile effect of NO in the systemic arteries in early postnatal ontogenesis (at the age of 11-14 days). Decreased anticontractile effect of NO can be the reason for insufficient blood supply and represent a risk factor for the development of cardiovascular disorders. IMPACT: The mechanisms of perinatal hypoxia influences on systemic circulation are almost unknown. We have shown that perinatal hypoxia weakens anticontractile influence of nitric oxide in early postnatal period. The influence of perinatal hypoxia on systemic circulation should be taken into account during treatment of newborns suffered from the lack of oxygen.

Effect of chronic administration of 17ß-estradiol on the vasopressor responses induced by the sympathetic nervous system in insulin resistance rats.

Several studies have demonstrated that the underlying mechanism of insulin resistance (IR) is linked with developing diseases like diabetes mellitus, hypertension, metabolic syndrome, and polycystic ovary syndrome. In turn, the dysfunction of female gonadal hormones (especially 17ß-estradiol) may be related to the development of IR complications since different studies have shown that 17ß-estradiol has a cardioprotector and vasorelaxant effect. This study aimed was to determine the effect of the 17ß-estradiol administration in insulin-resistant rats and its effects on cardiovascular responses in pithed rats. Thus, the vasopressor responses are induced by sympathetic stimulation or i.v. bolus injections of noradrenaline (α1/2), methoxamine (α1), and UK 14,304 (α2) adrenergic agonist were determined in female pithed rats with fructose-induced insulin resistance or control rats treated with: 1) 17ß-estradiol or 2) its vehicle (oil) for 5 weeks. Thus, 17ß-estradiol decreased heart rate, prevented the increase of blood pressure induced by ovariectomy, but with the opposite effect on sham-operated rats; and decreased vasopressor responses induced by i.v. bolus injections of noradrenaline on sham-operated (control and fructose group) and ovariectomized (control) rats, and those induced by i.v. bolus injections of methoxamine (α1 adrenergic agonist). Overall, these results suggest 17ß-estradiol has a cardioprotective effect, and its effect on vasopressor responses could be mediated mainly by the α1 adrenergic receptor. In contrast, IR with ovariectomy 17ß-estradiol decreases or loses its cardioprotector effect, this could suggest a possible link between the adrenergic receptors and the insulin pathway.

MAPKs Are Highly Abundant but Do Not Contribute to α1-Adrenergic Contraction of Rat Saphenous Arteries in the Early Postnatal Period.

Previously, the abundance of p42/44 and p38 MAPK proteins had been shown to be higher in arteries of 1- to 2-week-old compared to 2- to 3-month-old rats. However, the role of MAPKs in vascular tone regulation in early ontogenesis remains largely unexplored. We tested the hypothesis that the contribution of p42/44 and p38 MAPKs to the contraction of peripheral arteries is higher in the early postnatal period compared to adulthood. Saphenous arteries of 1- to 2-week-old and 2- to 3-month-old rats were studied using wire myography and western blotting. The α1-adrenoceptor agonist methoxamine did not increase the phosphorylation level of p38 MAPK in either 1- to 2-week-old or 2- to 3-month-old rats. Accordingly, inhibition of p38 MAPK did not affect arterial contraction to methoxamine in either age group. Methoxamine increased the phosphorylation level of p42/44 MAPKs in arteries of 2- to 3-month-old and of p44 MAPK in 1- to 2-week-old rats. Inhibition of p42/44 MAPKs reduced methoxamine-induced contractions in arteries of 2- to 3-month-old, but not 1- to 2-week-old rats. Thus, despite a high abundance in arterial tissue, p38 and p42/44 MAPKs do not regulate contraction of the saphenous artery in the early postnatal period. However, p42/44 MAPK activity contributes to arterial contractions in adult rats.

Inhibition of human prostate stromal cell growth and smooth muscle contraction by thalidomide: A novel remedy in LUTS?

BACKGROUND: Medical treatment in benign prostatic hyperplasia targets prostate size to prevent disease progression, complications, and surgery, and prostate smooth muscle tone for rapid relief of lower urinary tract symptoms. Combination therapies are still required to target both at once. However, current medications are insufficient, due to an unfavorable balance between side effects and efficacy. The limited efficacy of α1 -blockers may be due to nonadrenergic mediators like endothelin-1 and thromboxane A2 (TXA2 ), which keep up prostate smooth muscle contraction even in the presence of α1 -blockers. Consequently, future options with higher efficacy need to target α1 -adrenergic and nonadrenergic contractions as well as stromal cell growth at once. Thalidomide has been approved as an oral medication for various diseases, including the treatment of prostate cancer. Therefore, we investigated the effect of thalidomide on cellular functions of prostate stromal cells and human prostate smooth muscle contraction. METHODS: Cytoskeletal organization was visualized by phalloidin staining, cell growth was assessed by 5-ethynyl-2'-deoxyuridine assay, cell viability by cell counting kit-8, and apoptosis and cell death by flow cytometry in cultured prostate stromal cells (WPMY-1). Contractions of human prostate tissues from radical prostatectomy were studied in an organ bath, where they were induced by the α1 -adrenoceptor agonists methoxamine, noradrenaline, phenylephrine, and the nonadrenergic agonists endothelin-1, TXA2 analog U46619, or electric field stimulation (EFS). RESULTS: Thalidomide significantly reduced the proliferation of WPMY-1 cells, which was time- and concentration-dependent (10-300 µM). In parallel, organization of actin filaments collapsed after treatment with thalidomide. Thalidomide (30-100 µM) inhibited noradrenaline-, phenylephrine-, and methoxamine-induced contractions, as well as nonadrenergic contractions induced by endothelin-1 and U46619, and neurogenic contractions induced by EFS. No reduction in viability and no increases in apoptosis or in cell death were observed in WPMY-1 cells. CONCLUSIONS: Thalidomide impairs the growth of human prostate stromal cells, without showing a decrease in cell viability. In parallel, thalidomide inhibits adrenergic, neurogenic, and nonadrenergic contractions. This may be explained by a breakdown of the actin cytoskeleton. In vivo, urodynamic effects of thalidomide appear possible and may even exceed those of α1 -blockers or combination therapies.

Trophic sympathetic influence weakens pro-contractile role of Cl- channels in rat arteries during postnatal maturation.

Membrane transporters and their functional contribution in vasculature change during early postnatal development. Here we tested the hypothesis that the contribution of Cl- channels to arterial contraction declines during early postnatal development and this decline is associated with the trophic sympathetic influence. Endothelium-denuded saphenous arteries from 1- to 2-week-old and 2- to 3-month-old male rats were used. Arterial contraction was assessed in the isometric myograph, in some experiments combined with measurements of membrane potential. mRNA and protein levels were determined by qPCR and Western blot. Sympathectomy was performed by treatment with guanethidine from the first postnatal day until 8-9-week age. Cl- substitution in the solution as well as Cl--channel blockers (MONNA, DIDS) had larger suppressive effect on the methoxamine-induced arterial contraction and methoxamine-induced depolarization of smooth muscle cells in 1- to 2-week-old compared to 2- to 3-month-old rats. Vasculature of younger group demonstrated elevated expression levels of TMEM16A and bestrophin 3. Chronic sympathectomy increased Cl- contribution to arterial contraction in 2-month-old rats that was associated with an increased TMEM16A expression level. Our study demonstrates that contribution of Cl- channels to agonist-induced arterial contraction and depolarization decreases during postnatal development. This postnatal decline is associated with sympathetic nerves development.

Effects of agonists and phorbol esters on α1A-adrenergic receptor-Rab protein interactions.

In a cell line, stably expressing α1A-adrenoceptors fused to the mCherry red fluorescent protein, noradrenaline, methoxamine, and oxymetazoline induced concentration-dependent increases in intracellular calcium. All of these agents increase α1A-adrenoceptor phosphorylation and internalization. Transient co-expression of these receptors with Rab proteins tagged with the enhanced Green Fluorescent Protein was employed to estimate α1A-adrenoceptor-Rab interaction using Förster Resonance Energy Transfer. Noradrenaline and methoxamine increased α1A-adrenoceptor interaction with Rab5 and Rab7 but did not modify it with Rab9. Oxymetazoline induced adrenoceptor interaction with Rab5 and Rab9 and only an insignificant increase in Rab7 signal. Phorbol myristate acetate increased α1A-adrenoceptor interaction with Rab5 and Rab9 but did not modify it with Rab7. The agonists and the active phorbol ester, all of which induce receptor phosphorylation and internalization, favor receptor interaction with Rab5, i.e., association with early endosomes. Cell stimulation with phorbol myristate acetate induced the α1A-adrenoceptors to interact with the late endosomal marker, Rab9, suggesting that the receptors are directed to slow recycling endosomes once they have transited to the Trans-Golgi network to be retrieved to the plasma membrane. The agonists noradrenaline and methoxamine likely induce a faster recycling and might direct some of the adrenoceptors toward degradation and/or very slow recycling to the plasma membrane. Oxymetazoline produced a mixed pattern of interaction with the Rab proteins. These data indicate that α1A-adrenoceptor agonists can trigger different vesicular traffic and receptor fates within the cells.

Efficacy of vardenafil in human nasal mucosa.

OBJECTIVE: Levitra, a phosphodiesterase-5 (PDE5) inhibitor, is the trade name of vardenafil. It is applied to treatment of erectile dysfunction. PDE5 inhibitors dilate the penile blood vessels and cause prolonged erections. However, the effects of Levitra on human nasal mucosa are not yet fully explored. MATERIALS AND METHODS: We examined the effectiveness of Levitra on human nasal mucosa directly in vitro by testing: 1) effect on human nasal mucosa resting tension; 2) effect on contraction caused by 10-6 M methoxamine as a sympathetic mimetic; 3) effect of the drugs on electrically induced human nasal mucosa contractions. RESULTS: The results showed that addition of methoxamine to the incubation medium caused the nasal mucosa to contract in a dose-dependent manner. Addition of Levitra at doses of 10-4 M elicited a significant relaxation response to 10-6 M methoxamine-induced mucosa strip contraction. Levitra could not inhibit electrical field stimulation-induced spike contraction and had a minimal effect on the basal tension of nasal mucosa as the concentration increased. CONCLUSION: This study indicated that high concentrations of Levitra had a significant spasmolytic effect by antagonizing α-adrenoceptors. Moreover, nasal obstruction might not be relieved in patients suffering from erectile dysfunction and stuffy noses who were concomitant using α-adrenergic agonist and Levitra.

Augmentation index in the assessment of wave reflections and systolic loading.

BACKGROUND: Augmentation index (AIx) is used to quantify the augmented systolic aortic pressure that impedes ventricular ejection. Its use as an index of wave reflections is questionable. We hypothesize that AIx is quantitatively different from the reflection coefficient under varied physiological conditions. METHODS: 42 datasets of aortic pressure and flow waveforms were obtained during induced hypertension (methoxamine infusion) and vasodilation (nitroprusside infusion) in our mongrel dog experiments (n = 5) and from Mendeley data during various interventions (vasoconstrictors, vasodilators, pacing, stimulation, hemorrhage and hemodilution). Wave reflections and principal components of reflection coefficients were computed for comparison to AIx and heart rate normalized AIx. RESULTS: Principal reflection coefficient, Γ1, increased in hypertension and decreased in vasodilation, hemorrhage and hemodilution. AIx followed the trend in many cases but was consistently lower than Γ1 in almost all the subjects. The Bland-Altman analysis also showed that both AIx and normalized AIx underestimated Γ1. The relationship between augmentation index and reflection coefficient was explained by a linear regression model (r2 = 0.23, p < 0.01) in which AIx followed directional changes in Γ1 and the normalization of AIx resulted in a linear model that explained less variation in the relationship between AIx and Γ1. CONCLUSION: AIx is a reasonable clinical trend indicator, albeit not an accurate surrogate measure of the amount of wave reflections.

Potential vascular α1-adrenoceptor blocking properties of metformin in rat aorta and tail artery.

Metformin is a widely used drug for the treatment of type 2 Diabetes Mellitus. Several studies have also suggested that metformin decreases blood pressure; although an interaction with α-adrenoceptors has been proposed, this mechanism needs to be further investigated. Since α1-adrenoceptors play a significant role to regulate vascular tone, this study has analysed the potential ability of metformin to block α1-adrenoceptors in rat aorta and tail artery. For this purpose, the contractile responses induced by noradrenaline, methoxamine, and phenylephrine were determined in the absence or presence of metformin in rat aorta and tail artery rings. In both arteries, noradrenaline, methoxamine, and phenylephrine produced concentration-dependent contractile responses. Interestingly, the contractile responses to noradrenaline, methoxamine, and phenylephrine were significantly and differentially blocked by metformin (1, 3.1 and/or 10 mM) but not by vehicle. These results suggest that metformin is capable to block α1-adrenoceptors and may explain, at least in part, the anti-hypertensive effect observed in several clinical trials.

Effects of montelukast on human nasal mucosa.

OBJECTIVE: Montelukast is a selective and orally active leukotriene D4 receptor antagonist often used in treating asthma and allergic rhinitis. Montelukast nasal spray was developed to avoid systemic adverse effects of the drug in vitro. However, the effects of montelukast on human nasal mucosa are not yet fully explored and potential nasal vascular side effects of the drug merit further exploration. First, the effects of montelukast on vasocontractile responses generated by smooth muscles in the vascular structures of human nasal mucosa were investigated directly in vitro. METHODS: This study examined the effects of montelukast on human nasal mucosa in terms of mucosa resting tension, vasoconstriction caused by 10- 6 M methoxamine as a sympathetic mimetic, and electrically induced vasoconstrictions. RESULTS: The results indicated that addition of methoxamine to the incubation medium caused the nasal mucosa to vasocontract in a dose-dependent manner. Addition of montelukast at doses of 10- 5 M or above elicited a significant vasodilation response to 10- 6 M methoxamine-induced vasoconstriction. Montelukast could not inhibit electrical field stimulation-induced spike vasoconstriction. Moreover, increase in concentration of montelukast had minimal effect on basal tension of nasal mucosa. CONCLUSIONS: The study indicated significant vasodilation on human nasal mucosa under high concentrations of montelukast with a probable α-adrenoceptor antagonism. Hence, the nasal activity of α-adrenergic agonist nasal spray for nasal obstruction may be reduced in those using concomitant (oral or local spray) montelukast.

Pharmacological evidence that metformin blocks the vasopressor responses mediated by stimulation of α1- and α2-adrenoceptors in pithed rats.

It has been reported that metformin reduces blood pressure although the mechanisms have not been described. Indeed, several mechanisms could be implicated including the interaction with α-adrenoceptors or inhibition of sympathetic outflow. Therefore, this study was designed to determine the capability of metformin to block the vasopressor responses induced by α1/2-adrenoceptor agonists or selective electrical stimulation of sympathetic outflow. For this purpose, Wistar male rats were anesthetized, pithed and cannulated for selective preganglionic stimulation of the vasopressor sympathetic outflow or drugs administration. The effect of i.v. bolus injection of metformin (180 and 310mg/kg) or its vehicle (bidistilled water) was studied on the vasopressor responses induced by: (1) selective sympathetic stimulation (0.03-3Hz); (2) exogenous noradrenaline (0.03-3µg/kg); (3) methoxamine (1-100µg/kg); and (4) UK 14,304 (0.1-30µg/kg). The tachycardic responses to noradrenaline were also investigated in presence of metformin. The vasopressor responses induced by selective electrical stimulation of sympathetic outflow were diminished by metformin (180 and 310mg/kg) and remained unchanged in presence of vehicle. Moreover, the vasopressor responses induced by exogenous noradrenaline, methoxamine and UK 14,304 were dose-dependently inhibited by i.v. bolus injections of metformin (180 and 310mg/kg) and were not affected by vehicle. Metformin practically did not block the tachycardic responses to noradrenaline except at the dose of 3µg/kg. Taken together, these results demonstrate that metformin is capable to block vascular α1/2-adrenoceptors but not cardiac ß-adrenoceptors. Thus, this mechanism could contribute, at least in part, on the hypotensive responses induced by metformin.

Modulation of SK channels regulates locomotor alternating bursting activity in the functionally-mature spinal cord.

The spinal cord contains specialized groups of cells called pattern generators, which are capable of orchestrating rhythmic firing activity in an isolated preparation. Different patterns of activity could be generated in vitro including right-left alternating bursting and bursting in which both sides are synchronized. The cellular and network mechanisms that enable these behaviors are not fully understood. We have recently shown that Ca2+-activated K+ channels (SK channels) control the initiation and amplitude of synchronized bursting in the spinal cord. It is unclear, however, whether SK channels play a similar role in the alternating rhythmic pattern. In the current study, we used a spinal cord preparation from functionally mature mice capable of weight bearing and walking. The present results extend our previous work and show that SK channel inhibition initiates and modulates the amplitude of alternating bursting. We also show that addition of methoxamine, an α1-adrenergic agonist, to a cocktail of serotonin, dopamine, and NMDA evokes robust and consistent alternating bursting throughout the cord.

Adrenergic receptor agonists induce the differentiation of pluripotent stem cell-derived hepatoblasts into hepatocyte-like cells.

Current induction methods of hepatocytes from human induced pluripotent stem cells (hiPSCs) are neither low cost nor stable. By screening a chemical library of 1,120 bioactive compounds and known drugs, we identified the α1-adrenergic receptor agonist methoxamine hydrochloride as a small molecule that promotes the differentiation of hiPSC-derived hepatoblasts into ALBUMIN+ hepatocyte-like cells. Other α1-adrenergic receptor agonists also induced the differentiation of hepatocyte-like cells, and an α1-receptor antagonist blocked the hepatic-inducing activity of methoxamine hydrochloride and that of the combination of hepatocyte growth factor (HGF) and Oncostatin M (OsM), two growth factors often used for the induction of hepatoblasts into hepatocyte-like cells. We also confirmed that treatment with methoxamine hydrochloride activates the signal transducer and activator of transcription 3 (STAT3) pathway downstream of IL-6 family cytokines including OsM. These findings allowed us to establish hepatic differentiation protocols for both mouse embryonic stem cells (mESCs) and hiPSCs using small molecules at the step from hepatoblasts into hepatocyte-like cells. The results of the present study suggest that α1-adrenergic agonists induce hepatocyte-like cells by working downstream of HGF and OsM to activate STAT3.

Factors influencing biased agonism in recombinant cells expressing the human α1A -adrenoceptor.

BACKGROUND AND PURPOSE: Agonists acting at GPCRs promote biased signalling via Gα or Gßγ subunits, GPCR kinases and ß-arrestins. Since the demonstration of biased agonism has implications for drug discovery, it is essential to consider confounding factors contributing to bias. We have examined bias at human α1A -adrenoceptors stably expressed at low levels in CHO-K1 cells, identifying off-target effects at endogenous receptors that contribute to ERK1/2 phosphorylation in response to the agonist oxymetazoline. EXPERIMENTAL APPROACH: Intracellular Ca2+ mobilization was monitored in a Flexstation® using Fluo 4-AM. The accumulation of cAMP and ERK1/2 phosphorylation were measured using AlphaScreen® proximity assays, and mRNA expression was measured by RT-qPCR. Ligand bias was determined using the operational model of agonism. KEY RESULTS: Noradrenaline, phenylephrine, methoxamine and A61603 increased Ca2+ mobilization, cAMP accumulation and ERK1/2 phosphorylation. However, oxymetazoline showed low efficacy for Ca+2 mobilization, no effect on cAMP generation and high efficacy for ERK1/2 phosphorylation. The apparent functional selectivity of oxymetazoline towards ERK1/2 was related to off-target effects at 5-HT1B receptors endogenously expressed in CHO-K1 cells. Phenylephrine and methoxamine showed genuine bias towards ERK1/2 phosphorylation compared to Ca2+ and cAMP pathways, whereas A61603 displayed bias towards cAMP accumulation compared to ERK1/2 phosphorylation. CONCLUSION AND IMPLICATIONS: We have shown that while adrenergic agonists display bias at human α1A -adrenoceptors, the marked bias of oxymetazoline for ERK1/2 phosphorylation originates from off-target effects. Commonly used cell lines express a repertoire of endogenous GPCRs that may confound studies on biased agonism at recombinant receptors.

Retigabine diminishes the effects of acetylcholine, adrenaline and adrenergic agonists on the spontaneous activity of guinea pig smooth muscle strips in vitro.

PURPOSE: The aim of this study is to evaluate the effect of retigabine on the smooth muscle response to acetylcholine, adrenaline, α-and ß-adrenoceptor agonists. METHODS: We studied the change in the spontaneous smooth muscle contraction of guinea pig gastric corpus strips before and after 20-min treatment with 2µM retigabine. We also evaluated the effect of retigabine on the smooth muscle response to 10µM acetylcholine, 1 and 10µM adrenaline, 1µM methoxamine, 0.1µM p-iodoclonidine and 10µM isoproterenol. RESULTS: We observed a significant reduction in the effects of all studied mediators and agonists when they were added to organ baths in the presence of retigabine. Retigabine diminished the effect of acetylcholine on the spontaneous smooth muscle activity. The effect was fully antagonized by XE-991 (Kv7 channel blocker), which supports our hypothesis about the role of KCNQ channels in the registered changes. The increase in the contraction force after adding of 1µM adrenaline, methoxamine, and 0.1µM p-iodoclonidine was also significantly smaller in presence of retigabine. However, comparing the effect of 10µM adrenaline on the contractility before and after treatment with retigabine, we observed increased contractility when retigabine was present in the organ baths. CONCLUSION: A possible explanation for the observed diminished effects of mediators and receptor agonists is that the effect of retigabine on smooth muscle contractility is complex. The membrane hyperpolarization, the interaction between Kv7 channels and adrenoceptors, and the influence on signaling pathways may contribute to the summary smooth muscle response.

Shaping the Output of Lumbar Flexor Motoneurons by Sacral Neuronal Networks.

The ability to improve motor function in spinal cord injury patients by reactivating spinal central pattern generators (CPGs) requires the elucidation of neurons and pathways involved in activation and modulation of spinal networks in accessible experimental models. Previously we reported on adrenoceptor-dependent sacral control of lumbar flexor motoneuron firing in newborn rats. The current work focuses on clarification of the circuitry and connectivity involved in this unique modulation and its potential use. Using surgical manipulations of the spinal gray and white matter, electrophysiological recordings, and confocal microscopy mapping, we found that methoxamine (METH) activation of sacral networks within the ventral aspect of S2 segments was sufficient to produce alternating rhythmic bursting (0.15-1 Hz) in lumbar flexor motoneurons. This lumbar rhythm depended on continuity of the ventral funiculus (VF) along the S2-L2 segments. Interrupting the VF abolished the rhythm and replaced it by slow unstable bursting. Calcium imaging of S1-S2 neurons, back-labeled via the VF, revealed that ∼40% responded to METH, mostly by rhythmic firing. All uncrossed projecting METH responders and ∼70% of crossed projecting METH responders fired with the concurrent ipsilateral motor output, while the rest (∼30%) fired with the contralateral motor output. We suggest that METH-activated sacral CPGs excite ventral clusters of sacral VF neurons to deliver the ascending drive required for direct rhythmic activation of lumbar flexor motoneurons. The capacity of noradrenergic-activated sacral CPGs to modulate the activity of lumbar networks via sacral VF neurons provides a novel way to recruit rostral lumbar motoneurons and modulate the output required to execute various motor behaviors. SIGNIFICANCE STATEMENT: Spinal central pattern generators (CPGs) produce the rhythmic output required for coordinating stepping and stabilizing the body axis during movements. Electrical stimulation and exogenous drugs can reactivate the spinal CPGs and improve the motor function in the absence of descending supraspinal control. Since the body-stabilizing sacral networks can activate and modulate the limb-moving lumbar circuitry, it is important to clarify the functional organization of sacral and lumbar networks and their linking pathways. Here we decipher the ascending circuitry linking adrenoceptor-activated sacral CPGs and lumbar flexor motoneurons, thereby providing novel insights into mechanisms by which sacral circuitry recruits lumbar flexors, and enhances the motor output during lumbar afferent-induced locomotor rhythms. Moreover, our findings might help to improve drug/electrical stimulation-based therapy to accelerate locomotor-based rehabilitation.

Peculiar Aspects in Influence of α1-Adrenoceptor Stimulation on Isolated Rat Heart.

The study examined the effect of α1-adrenoceptor stimulation with methoxamine on chronotropic function of isolated heart perfused ex vivo according to Langendorff and cardiac chronotropy in vivo. Stimulation of α1-adrenoceptors in isolated heart induced gradually developing bradycardia, which progressed during several minutes. Similar stimulation in vivo produced a short-term bradycardia probably terminated by the compensatory influences in the whole organism. Comparison of the data obtained in both experimental paradigms during α1-adrenoceptor stimulation revealed unidirectional changes in cardiac chronotropy characterized with time-related peculiarities.

Kcne4 Deletion Sex-Dependently Alters Vascular Reactivity.

Voltage-gated potassium (Kv) channels formed by Kv7 (KCNQ) α-subunits are recognized as crucial for vascular smooth muscle function, in addition to their established roles in the heart (Kv7.1) and the brain (Kv7.2-5). In vivo, Kv7 α-subunits are often regulated by KCNE subfamily ancillary (ß) subunits. We investigated the effects of targeted germline Kcne4 deletion on mesenteric artery reactivity in adult male and female mice. Kcne4 deletion increased mesenteric artery contractility in response to α-adrenoceptor agonist methoxamine, and decreased responses to Kv7.2-7.5 channel activator ML213, in male but not female mice. In contrast, Kcne4 deletion markedly decreased vasorelaxation in response to isoprenaline in both male and female mice. Kcne4 expression was 2-fold lower in the female versus the male mouse mesenteric artery, and Kcne4 deletion elicited only moderate changes of other Kcne transcripts, with no striking sex-specific differences. However, Kv7.4 protein expression in females was twice that in males, and was reduced in both sexes by Kcne4 deletion. Our findings confirm a crucial role for KCNE4 in regulation of Kv7 channel activity to modulate vascular tone, and provide the first known molecular mechanism for sex-specificity of this modulation that has important implications for vascular reactivity and may underlie sex-specific susceptibility to cardiovascular diseases.

The calcilytics Calhex-231 and NPS 2143 and the calcimimetic Calindol reduce vascular reactivity via inhibition of voltage-gated Ca2+ channels.

The present study investigates the effect of commonly used negative and positive allosteric modulators of the calcium-sensing receptor (CaSR) on vascular reactivity. In wire myography studies, increasing [Ca2+]o from 1mM to 6mM induced concentration-dependent relaxations of methoxamine-induced pre-contracted rabbit mesenteric arteries, with 6mM [Ca2+]o producing almost complete relaxation. [Ca2+]o-induced relaxations were attenuated in the presence of the calcilytics Calhex-231 and NPS 2143, and abolished by the removal of the endothelium. In addition to their calcilytic effects, Calhex-231 and NPS 2143 also produced concentration-dependent inhibitions of methoxamine- or KCl-induced precontracted tone, which were unaffected by removal of the endothelium and unopposed in the presence of the calcimimetic Calindol. In vessels with depleted Ca2+ stores, contractions mediated by Ca2+ influx via voltage-gated Ca2+ channels (VGCCs) were inhibited by Calhex231. In freshly isolated single rabbit mesenteric artery smooth muscle cells, Calhex-231 and NPS 2143 inhibited whole-cell VGCC currents. Application of Calindol also inhibited methoxamine- and KCl-induced pre-contracted tone, and inhibited whole-cell VGCC currents. In conclusion, in addition to their CaSR-mediated actions in the vasculature, Calhex-231, NPS 2143 and Calindol reduce vascular contractility via direct inhibition of VGCCs.