Effects of urocortin on T-type calcium currents in mouse spermatogenic cells

doi:10.1016/j.bbrc.2005.02.041

Biochemical and Biophysical Research Communications

Volume 329, Issue 2, 8 April 2005, Pages 743-748

https://doi.org/10.1016/j.bbrc.2005.02.041 Get rights and content

Abstract

Urocortin (UCN), a newly isolated peptide, has been found to play an important role mainly in female reproductive system. In order to investigate the effect of UCN on T-type calcium currents (I_Ca,T), exploring the mechanisms of UCN’s role in male reproductive system, especially in acrosome reaction, we directly measured the I_Ca,T in mouse spermatogenic cells exposed to UCN using standard whole-cell patch-clamp recording technique. Our results showed that UCN reversibly inhibited the T-type Ca²⁺ currents in the cells in a concentration-dependent manner. The current density was inhibited by about 19% after exposure of the cells to UCN (0.1 μM) for 5 min, from the control value of 6.75 ± 1.17 to 5.26 ± 0.82 pA/pF. UCN up-shifted the current–voltage (I–V) curve. Frequency-dependence of UCN’s effects on I_Ca,T was also observed. Moreover, UCN at 0.1 μM did not markedly affect the activation of I_Ca,T but shifted the inactivation curve of I_Ca,T to the left. The inhibitory effect of UCN on the T-type Ca²⁺ current was not affected by Astressin, the CRF receptor blocker. Since T-type calcium channels are a key component in acrosome reaction, our data suggest that UCN might be a significant factor in male reproductive action and a potential contraceptive agent.

Section snippets

Materials and methods

These studies were carried out in agreement with the official recommendations of The Chinese Community guidelines.

Solutions. The composition of the external solution for T-type calcium current recording was composed of (in mM): 10 CaCl₂, 130 NaCl, 3 KCl, 2 MgCl₂, 1 NaHCO₃, 0.5 NaH₂PO₄, 5 HEPES, and 10 glucose, pH adjusted with NaOH to 7.3. The pipette solution contained (in mM): 100 CsCl, 10 CsF, 5 EGTA, 5 HEPES, 4 Mg–ATP, and 4 phosphocreatine, pH adjusted with CsOH to 7.3 [21]. The culture

Results

As shown in Fig. 1A, a series of evoked current traces were recorded when a spermatogenic cell was given designed depolarizing test pulses. There were few steady state components in the current. The current was not affected by 1 μM Bay K8644, a specific L-type calcium channel activator (n = 3, Fig. 1B). After perfusion with 200 μM Ni²⁺-containing solution, the current was drastically inhibited (n = 3, Fig. 1C). The low voltage-activated, fast inactivating, steady state component-free and Ni²⁺

Discussion

Reproduction is a complex process, whose mechanisms are being explored worldwide by scientists. Recently, UCN was found to exist in the reproductive system, especially in seminal fluid [16]. These interesting reports lead to our hypothesis that UCN might have a significant effect on male reproductive functions such as acrosome reactions and capacitation which are supposed to be associated with T-type calcium channels.

As mentioned above, UCN possesses a wide spectrum of physiological and

Acknowledgments

We thank Dr. Xiangjian Chen for her help in the statistical analysis. This work was supported by Ministry of Education Returning Overseas Scholar Science Study Foundation and Qinglan Plan of Jiangsu Province.

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Although the protein tyrosine kinase (PTK) inhibitor, genistein, has been widely used to investigate the possible involvement of PTK during reproductive functions, it is unknown whether it modulates sperm calcium channel activity. In the present study, we recorded T-type calcium currents (I_Ca,T) in mouse spermatogenic cells using whole-cell patch clamp and found that extracellular application of genistein reversibly decreased I_Ca,T in a concentration-dependent manner (IC₅₀ ∼22.7 μM). To determine whether TK activity is required for I_Ca,T inhibition, we found that peroxovanadate, a tyrosine phosphatase inhibitor, was ineffective in preventing the inhibitory effect of genistein. Furthermore, intracellular perfusion of the cells with ATP-γ-S also did not alter the inhibitory effect of genistein. To further reveal the direct inhibitory mechanism of genistein on I_Ca,T, we applied into the bath lavendustin A, a PTK inhibitor structurally unrelated to genistein, and found that the current amplitude remained unchanged. Moreover, daidzein, an inactive structural analog of genistein, robustly inhibited the currents. The inhibitory effect of genistein on T-type calcium channels was associated with a hyperpolarizing shift in the voltage-dependence of inactivation. Genistein was observed to decrease sperm motility and to significantly inhibit sperm acrosome reaction (AR) evoked by zona pellucida. Using transfected HEK293 cells system, only Cav3.1 and Cav3.2, instead of Cav3.3, channels were inhibited by genistein. Since T-type calcium channels are the key components in the male reproduction, such as in AR and sperm motility, our data suggest that this PTK-independent inhibition of genistein on I_Ca,T might be involved in its anti-reproductive effects.
Modulation of T-type Ca<sup>2+</sup> channels by corticotropin-releasing factor through protein kinase C pathway in MN9D dopaminergic cells
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Changes of Ca2+ signals by CRF were reported in several systems. Urocortin inhibits L-type Ca2+ channels from acute isolated adult rat ventricular cells [25], and directly reduces T-type Ca2+ currents with receptor-independent manner in mouse spermatogenic cells [26]. Urocortin 2 also reduces the intracellular Ca2+ levels via inhibiting L-type Ca2+ channel in PC12 cells [27].
Corticotrophin-releasing factor (CRF) is the main regulator of the body’s stress axis and its signal is translated through G-protein-coupled CRF receptors (CRF-R1, CRF-R2). Even though CRF receptors are present in the midbrain dopamine neurons, the cellular mechanism of CRF action is not clear yet. Since voltage-dependent Ca²⁺ channels are highly expressed and important in dopamine neuronal functions, we tested the effect of CRF on voltage-dependent Ca²⁺ channels in MN9D cells, a model of dopamine neurons. The application of CRF-related peptide, urocortin 1, reversibly inhibited T-type Ca²⁺ currents, which was a major Ca²⁺ channel in the cells. The effect of urocortin was abolished by specific CRF-R1 antagonist and was mimicked by protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate. PKC inhibitors abolished the effect of urocortin. These results suggest that urocortin modulates T-type Ca²⁺ channel by interacting with CRF-R1 via the activation of PKC signal pathway in MN9D cells.
T-type Ca<sup>2+</sup> channels in sperm function
2006, Cell Calcium
Citation Excerpt :
Mouse spermatogenic cells mainly display CaV3 currents with pharmacological characteristics resembling those of the initial Ca2+ uptake that occurs during the ZP-induced AR. Recently developed inhibitors of these CaV currents such as urocortin, fenvalerate and gossypol may have contraceptive applications [24,66,67]. These observations, taken together indicate that T-type Ca2+ channels may participate in the mouse AR [12,19,23].
Intracellular Ca²⁺ regulates many fundamental physiological processes in excitable and non-excitable cells. Certainly this is the case of sperm where the local concentration of intracellular Ca²⁺ ([Ca²⁺]i) is significantly influenced by Ca²⁺ permeable channels present in the cell plasma membrane. Amongst these channels, the voltage dependent Ca²⁺ channels (Ca_V) of the T-type (Ca_V3) appear to have an eminent role in the acrosome reaction (AR) of some sperm species, though they may participate in other important functions like motility and capacitation. The AR is an exocytotic event where the acrosome vesicle in the posterior region of the head fuses with the plasma membrane. This reaction allows sperm to fuse and fertilize the egg. Here we summarize our present knowledge regarding Ca_V3 channels in sperm, show the first direct electrophysiological evidence for their presence in maturing mouse sperm and discuss some of the relevant unanswered questions.
Enhanced expression of urocortin in lung tissues of rats with allergic asthma
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The expression of CRH-R2 has been detected in the lungs [42]. Other recent studies reported that UCN may play biologically diverse roles by acting on the ion channel directly but not CRHR [43–46]. The effect of UCN on asthmatic pulmonary inflammation may be mediated by activation of CRH receptors or by the ionic mechanisms.
Bronchial asthma is defined as a chronic airway inflammatory disease characterized by sustained activation of many inflammatory cells including mast cells. Urocortin (UCN) is synthesized and secreted by human mast cells and activated mast cells release more UCN. On the other hand, UCN can induce mast cell degranulation and generation of many proinflammatory factors. The purpose of this study was to examine the expression profile of UCN in rat lung with allergic asthma. Twenty-four male Sprague–Dawley rats were allocated to normal control, asthma model, and dexamethasone group, respectively. Animals were actively sensitized by subcutaneous injection of ovalbumin (OVA) and challenged by an aerosol of 1% OVA 2 weeks after sensitization. Both UCN mRNA and peptide were expressed in normal rat lungs. Rats in asthma model group developed severe infiltration of inflammatory cells and inflammation in airway, together with a significantly up-regulated expression of urocortin mRNA detected by semi-quantitative reverse transcriptase-polymerase chain reaction and peptide measured both by immunohistochemistry and Western blot analysis. In contrast, treatment with dexamethasone resulted in markedly ameliorated airway inflammation and alleviated airway inflammatory cell infiltration, coupled with a significantly decreased urocortin expression. Regression analysis revealed a positive correlation between urocortin expression and the number of inflammatory cells in bronchoalveolar lavage fluid (P < 0.01). In the present study, we first demonstrated that UCN was locally produced in rat lungs and expressed more pronouncedly in inflammatory airway of asthmatic rats. Glucocorticoid treatment markedly reduced the production of UCN in asthmatic lung tissues. Peripherally produced UCN in lung may act as a possible local autocrine and paracrine immune-inflammatory mediator in inflammatory airway of allergic asthma rats.
Urocortin reduces the viability of adult rat vascular smooth muscle cells via inhibiting L-type calcium channels
2005, Peptides
The newly isolated peptide, urocortin (UCN), is a member of the corticotropin-releasing factor (CRF)-related peptides that has been found to have potent cardiovascular protective effects. In order to investigate the effect of UCN on the viability of adult rat vascular smooth muscle cells (VSMC) and the relevant mechanisms, we exposed the VSMC to UCN to observe the change in cell viability using MTT assay and intracellular calcium concentration using confocal laser scanning microscope methods. Our results showed that UCN (10⁻⁷ M) inhibited the viability of VSMC by about 26% (P < 0.05, compared to control). The effect was concentration-dependent, but it was not dependent on the affecting time. Glybenclamide (Gly, 10⁻⁵ M), the ATP-sensitive potassium channel (K_ATP channel) blocker, and astressin (10⁻⁶ M), a competitive antagonist of CRF receptors, had no influence on this inhibition. Bay K8644 (10⁻⁶ M), a special L-type calcium channel activator, increased the viability of VSMC. Pre-treatment of the cells with UCN diminished the effect of Bay K8644 (n = 6, P < 0.05). UCN was also observed to reduce the intracellular Ca²⁺ increase induced by KCl and Bay K8644. There was no significant difference in nitrite accumulation between UCN groups and the control. In conclusion, UCN reduced the viability of VSMC through L-type calcium channels. These interesting results might suggest that UCN may be a new vasoactive agent involved in hindering vascular remodeling in combination with previous reports about UCN's hypotensive effects.
Effects of urocortin via ion mechanisms or CRF receptors?
2005, Biochemical and Biophysical Research Communications
Citation Excerpt :
Inhibition of Ca2+ influx could shorten action potential duration (APD) [26], resulting in a cardioprotective action due to decrease in energy consumption via the preservation of high-energy phosphates [27,28]. In male reproductive system, we also found that UCN could reduce the T-type calcium currents in mouse spermatogenic cells directly via inhibiting the calcium channels also instead of binding CRF-R2 receptors (Fig. 2) [29]. These novel results highlight the effect of UCN on male reproductive functions such as acrosome reactions and capacitation, which were supposed to be associated with calcium channels.
Urocortin (UCN), a newly isolated peptide related to hypothalamic corticotrophin releasing factor (CRF) family, had been reported to play biologically diverse roles in several systems such as cardiovascular, reproductive, appetite, stress, and inflammatory responses, etc. It was thought previously to be an endogenous agonist, producing the several actions previously attributed to CRF. But, recently, it was shown to directly reduce L-type calcium currents of acute isolated cardiac myocytes and T-type calcium currents in mouse spermatogenic cells via inhibiting calcium channel instead of binding first to its CRF-R₂ receptors. UCN could also reduce the intracellular calcium in vascular smooth muscle cells via inhibiting calcium channel directly. Furthermore, UCN could increase the gene expression of ATP-sensitive potassium channels (K_ATP) and activate sarcolemmal ATP-sensitive potassium current during normal or hypoxia, which could be inhibited by glibenclamide, a specific K_ATP blocker. This review will highlight the current novel findings on the ionic mechanisms by which UCN may exert its several actions.

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Effects of urocortin on T-type calcium currents in mouse spermatogenic cells

Abstract

Section snippets

Materials and methods

Results

Discussion

Acknowledgments

Genomics

Peptides

Neuropeptides

Peptides

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

FEBS Lett.

FEBS Lett.

Eur. J. Pharmacol.

Dev. Biol.

Pharmacol. Res.

Urocortin, a mammalian neuropeptide related to fish urotensin I and to corticotrophin-releasing factor

Nature

Cloning and characterization of human urocortin

Endocrinology

Human lymphocytes produce UCN, but not corticotrophin releasing hormone

J. Clin. Endocrinol. Metab.