Heat-inactivated Lactobacillus plantarum nF1 promotes intestinal health in Loperamide-induced constipation rats.

Constipation is a common condition that affects individuals of all ages, and prolonged constipation needs to be prevented to avoid potential complications and reduce the additional stress on individuals with pre-medical conditions. This study aimed to evaluate the effects of heat-inactivated Lactobacillus plantarum (HLp-nF1) on loperamide-induced constipation in rats. Constipation-induced male rats were treated orally with low to high doses of HLp-nF1 and an anti-constipation medication Dulcolax for five weeks. Study has 8 groups, control group; loperamide-treated group; Dulcolax-treated group; treatment with 3.2 × 1010, 8 × 1010 and 1.6 × 1011, cells/mL HLp-nF1; Loperamide + Dulcolax treated group. HLp-nF1 treated rats showed improvements in fecal pellet number, weight, water content, intestinal transit length, and contractility compared to the constipation-induced rats. Also, an increase in the intestine mucosal layer thickness and the number of mucin-producing crypt epithelial cells were observed in HLp-nF1-treated groups. Further, the levels of inflammatory cytokines levels were significantly downregulated by treatment with HLp-nF1 and Dulcolax. Notably, the metagenomics sequencing analysis demonstrated a similar genus pattern to the pre-preparation group and control with HLp-nF1 treatment. In conclusion, the administration of >3.2 × 1010 cells/mL HLp-nF1 has a positive impact on the constipated rats overall health.

Melatonin Suppresses the Kainate Receptor-Mediated Excitation on Gonadotropin-Releasing Hormone Neurons in Female and Male Prepubertal Mice.

Melatonin, a pineal gland secretion, is an amphiphilic neurohormone involved in the biological and physiologic regulation of bodily functions. Numerous studies have shown the effects of melatonin on the release of gonadotropins and their actions at one or several levels of the hypothalamic-pituitary-gonadal axis. However, direct melatonin action on gonadotropin-releasing hormone (GnRH) neurons and its mechanism of action remain unclear. Here, plasma melatonin levels were measured and the effect of melatonin on GnRH neurons was assessed using brain slice patch clamp techniques. The plasma melatonin levels in prepubertal mice were higher than those in the adults. Melatonin itself did not change the firing activity of GnRH neurons. Interestingly, the kainate receptor-mediated responses but not the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)- and N-methyl-D-aspartic acid (NMDA)-induced responses were suppressed by melatonin in both the voltage clamp and current clamp modes. The inhibitory effects of the kainate-induced response by melatonin tended to increase with higher melatonin concentrations and persisted in the presence of tetrodotoxin, a voltage-sensitive Na+ channel blocker, or luzindole, a non-selective melatonin receptor antagonist. However, the response was completely abolished by pretreatment with pertussis toxin. These results suggest that melatonin can regulate GnRH neuronal activities in prepubertal mice by partially suppressing the excitatory signaling mediated by kainate receptors through pertussis toxin-sensitive G-protein-coupled receptors.

Protein tyrosine phosphatase 1B is a mediator of cyclic ADP ribose-induced Ca2+ signaling in ventricular myocytes.

Cyclic ADP-ribose (cADPR) releases Ca2+ from ryanodine receptor (RyR)-sensitive calcium pools in various cell types. In cardiac myocytes, the physiological levels of cADPR transiently increase the amplitude and frequency of Ca2+ (that is, a rapid increase and decrease of calcium within one second) during the cardiac action potential. In this study, we demonstrated that cADPR levels higher than physiological levels induce a slow and gradual increase in the resting intracellular Ca2+ ([Ca2+]i) level over 10 min by inhibiting the sarcoendoplasmic reticulum Ca2+ ATPase (SERCA). Higher cADPR levels mediate the tyrosine-dephosphorylation of α-actin by protein tyrosine phosphatase 1B (PTP1B) present in the endoplasmic reticulum. The tyrosine dephosphorylation of α-actin dissociates phospholamban, the key regulator of SERCA, from α-actin and results in SERCA inhibition. The disruption of the integrity of α-actin by cytochalasin B and the inhibition of α-actin tyrosine dephosphorylation by a PTP1B inhibitor block cADPR-mediated Ca2+ increase. Our results suggest that levels of cADPR that are relatively higher than normal physiological levels modify calcium homeostasis through the dephosphorylation of α-actin by PTB1B and the subsequent inhibition of SERCA in cardiac myocytes.

Seminal CD38 is a pivotal regulator for fetomaternal tolerance.

A successful pregnancy depends on a complex process that establishes fetomaternal tolerance. Seminal plasma is known to induce maternal immune tolerance to paternal alloantigens, but the seminal factors that regulate maternal immunity have yet to be characterized. Here, we show that a soluble form of CD38 (sCD38) released from seminal vesicles to the seminal plasma plays a crucial role in inducing tolerogenic dendritic cells and CD4(+) forkhead box P3(+) (Foxp3(+)) regulatory T cells (Tregs), thereby enhancing maternal immune tolerance and protecting the semiallogeneic fetus from resorption. The abortion rate in BALB/c females mated with C57BL/6 Cd38(-/-) males was high compared with that in females mated with Cd38(+/+) males, and this was associated with a reduced proportion of Tregs within the CD4(+) T-cell pool. Direct intravaginal injection of sCD38 to CBA/J pregnant mice at preimplantation increased Tregs and pregnancy rates in mice under abortive sonic stress from 48 h after mating until euthanasia. Thus, sCD38 released from seminal vesicles to the seminal plasma acts as an immunoregulatory factor to protect semiallogeneic fetuses from maternal immune responses.

Percutaneous transplantation of human umbilical cord-derived mesenchymal stem cells in a dog suspected to have fibrocartilaginous embolic myelopathy.

The use of human umbilical cord blood-derived mesenchymal stem cells for cell transplantation therapy holds great promise for repairing spinal cord injury. Here we report the first clinical trial transplantation of human umbilical cord (hUCB)-derived mesenchymal stem cells (MSCs) into the spinal cord of a dog suspected to have fibrocartilaginous embolic myelopathy (FCEM) and that experienced a loss of deep pain sensation. Locomotor functions improved following transplantation in a dog. Based on our findings, we suggest that transplantation of hUCB-derived MSCs will have beneficial therapeutic effects on FCEM patients lacking deep pain sensation.

Dendroaspis natriuretic peptide regulates the cardiac L-type Ca2+ channel activity by the phosphorylation of α1c proteins.

Dendroaspis natriuretic peptide (DNP), a new member of the natriuretic peptide family, is structurally similar to atrial, brain, and C-type natriuretic peptides. However, the effects of DNP on the cardiac function are poorly defined. In the present study, we examined the effect of DNP on the cardiac L-type Ca(2+) channels in rabbit ventricular myocytes. DNP inhibited the L-type Ca(2+) current (I(Ca,L)) in a concentration dependent manner with a IC(50) of 25.5 nM, which was blocked by an inhibitor of protein kinase G (PKG), KT5823 (1 µM). DNP did not affect the voltage dependence of activation and inactivation of I(Ca,L). The α(1c) subunit of cardiac L-type Ca(2+) channel proteins was phosphorylated by the treatment of DNP (1 µM), which was completely blocked by KT5823 (1 µM). Finally, DNP also caused the shortening of action potential duration in rabbit ventricular tissue by 22.3 ± 4.2% of the control (n = 6), which was completely blocked by KT5823 (1 µM). These results clearly indicate that DNP inhibits the L-type Ca(2+) channel activity by phosphorylating the Ca(2+) channel protein via PKG activation.

Magnesium increases iberiotoxin-sensitive large conductance calcium activated potassium currents on the basilar artery smooth muscle cells in rabbits.

OBJECTIVES: Magnesium has been known for treating vasospasm following subarachnoid hemorrhage. However, its action mechanism in cerebral vascular relaxation is not clear. Potassium channels play a pivotal role in the relaxation of smooth muscle cells. To investigate their role in magnesium-induced relaxation of basilar smooth muscle cells, we examined the effect of magnesium on potassium channels using the patch clamp technique on acutely isolated smooth muscle cells from rabbit basilar artery. METHOD: Fresh smooth muscle cells were isolated from the basilar artery by enzyme treatment. To identify which potassium channels are involved in the magnesium-induced currents, we used the potassium channel blockers tetraethylammonium (TEA), glibenclamide, apamin and iberiotoxin (IBX). RESULTS: Magnesium (5 mM) increased the step pulse-induced outward K+ currents by 46% over control level (P < 0.01). The outward K+ current was decreased to 22% (P < 0.01) by TEA (10 mM), a non-specific K+ channel blocker, and to 60% of control level (P < 0.01) by IBX (0.1 µM,), a large-conductance Ca2+-activated K+ (BK) channel blocker, but was not inhibited by apamin (1 nM), a small-conductance Ca2+ -activated potassium (SK) channel blocker, or glibenclamide (3 mM), an adenosine triphosphate (ATP)-sensitive K+) channel blocker. Caffeine (3 mM) enhanced outward K+ currents. Magnesium-induced increase of outward K+ currents persisted in the presence of apamin. However, magnesium failed to increase the outward K+ currents in the presence of IBX. DISCUSSION: These results demonstrate that BK channels are functionally expressed in rabbit basilar smooth muscle cells and suggest that BK channels may play a pivotal role in magnesium-induced relaxation.

Tonic extrasynaptic GABA(A) receptor currents control gonadotropin-releasing hormone neuron excitability in the mouse.

It is well established that the GABA(A) receptor plays an important role in regulating the electrical excitability of GnRH neurons. Two different modes of GABA(A) receptor signaling exist: one mediated by synaptic receptors generating fast (phasic) postsynaptic currents and the other mediated by extrasynaptic receptors generating a persistent (tonic) current. Using GABA(A) receptor antagonists picrotoxin, bicuculline methiodide, and gabazine, which differentiate between phasic and tonic signaling, we found that ∼50% of GnRH neurons exhibit an approximately 15-pA tonic GABA(A) receptor current in the acute brain slice preparation. The blockade of either neuronal (NO711) or glial (SNAP-5114) GABA transporter activity within the brain slice revealed the presence of tonic GABA signaling in ∼90% of GnRH neurons. The GABA(A) receptor δ subunit is only found in extrasynaptic GABA(A) receptors. Using single-cell RT-PCR, GABA(A) receptor δ subunit mRNA was identified in GnRH neurons and the δ subunit-specific agonist 4,5,6,7-tetrahydroisoxazolo [5,4-c] pyridin-3-ol was found to activate inward currents in GnRH neurons. Perforated-patch clamp studies showed that 4,5,6,7-tetrahydroisoxazolo [5,4-c] pyridin-3-ol exerted the same depolarizing or hyperpolarizing effects as GABA on juvenile and adult GnRH neurons and that tonic GABA(A) receptor signaling regulates resting membrane potential. Together, these studies reveal the presence of a tonic GABA(A) receptor current in GnRH neurons that controls their excitability. The level of tonic current is dependent, in part, on neuronal and glial GABA transporter activity and mediated by extrasynaptic δ subunit-containing GABA(A) receptors.

Somatostatin inhibition of gonadotropin-releasing hormone neurons in female and male mice.

Previous studies indicate that somatostatin regulates gonadotropin secretion. We investigated here whether somatostatin has direct effects on GnRH neurons in the adult male and female mice. Dual-labeling immunofluorescence experiments revealed the presence of somatostatin-immunoreactive fibers adjacent to GnRH neurons, and three-dimensional confocal reconstructions demonstrated apparent somatostatin fiber appositions with 50-60% of GnRH neurons located throughout the brain in both male and female mice. Perforated patch-clamp recordings from GnRH-green fluorescent protein neurons revealed that approximately 70% of GnRH neurons responded in a dose-dependent manner to 10-300 nm somatostatin with an acute membrane hyperpolarization and cessation of firing. This effect persisted in the presence of tetrodotoxin and amino acid receptor antagonists, indicating a direct postsynaptic site of action on the GnRH neuron. The identity of the somatostatin receptors underlying this action was assessed using GnRH neuron single-cell RT-PCR. Of the somatostatin receptor subtypes, the sstr2 transcript was the most prevalent and detected in both males and females. The expression of sstr2 by GnRH neurons was confirmed in the sstr2 knockout/LacZ knock-in mouse line. Electrophysiological studies demonstrated that the sstr2-selective agonist seglitide exerted acute hyperpolarizing actions on GnRH neurons identical to those of somatostatin. Together, these studies reveal somatostatin, acting through sstr2, to be one of the most potent inhibitors of electrical excitability of male and female GnRH neurons identified thus far.