J Korean Med Sci.  2005 Apr;20(2):256-261. 10.3346/jkms.2005.20.2.256.

M3 Subtype of Muscarinic Receptors Mediate Ca2+ Release from Intracellular Stores in Rat Prostate Neuroendocrine Cells

• Kim JK
• Kim SJ
• Lee JE
• Min KM
• Kim SJ

• KMID: 1095200
• DOI: http://doi.org/10.3346/jkms.2005.20.2.256

Abstract

Our previous studies document the expression of adrenoceptors and purinoceptors in the rat prostate neuroendocrine cells (RPNECs). However, a direct investigation of the receptors for acetylcholine (ACh) is still lacking in the prostate neuroendocrine cells. RPNECs were freshly isolated from the ventral lobes of rat prostate by using collagenase. Effects of ACh and various muscarinic antagonists on the intracellular Ca2+ concentration ([Ca2+]c ) were investigated by using the fura-2 spectrofluorimetry. Single-cell RT-PCR analysis was applied to identify the transcripts for the muscarinic receptor subtypes. ACh (5 micrometer) induced a sharp transient increase in the [Ca2+]c of RPNECs, which was independent of the extracellular Ca2+. In the same RPNECs, high KCl (60 mM), phenylephrine (5micrometer), UTP (P2Y1/2 agonist, 50, micrometer), and alpha, beta-meATP (P2X1/3 agonist, 0.5micrometer) also increased the [Ca2+]c. The ACh-induced [Ca2+]c change (delta[Ca2+]c ) was blocked by atropine or by para-fluorohexahydrosiladifenidol (M3 antagonist, 0.3micrometer), but not by telenzepine (M1 antagonist, 1 micrometer) and himbacine (M2 and M4 antagonist, 1 mircoM). The single-cell RT-PCR demonstrated the selective expression of mRNAs for M3 in RPNECs. In summary, RPNECs express M3 muscarinic receptors that are linked to the release of Ca2+ from intracellular stores. The Ca2+ signals of RPNECs might mediate the parasympathetic regulation of prostate gland.

Keyword

Prostate; Neurosecretory Systems; Receptor, Muscarinic M3; Calcium Signaling; Rat; Fura-2

MeSH Terms

Acetylcholine/pharmacology
Animals
Calcium/*metabolism
Calcium Signaling
Male
Neurosecretory Systems/*metabolism
Prostate/*metabolism
Rats
Rats, Sprague-Dawley
Receptor, Muscarinic M3/*physiology
Research Support, Non-U.S. Gov't

Figure

• 

  Fig. 1 Ca2+ responses to various agonists in RPNECs. (A) representative traces of F340/380 demonstrating the effects of PhE (5 µM), UTP (20 µM), ACh (5 µM) and α,β-meATP (0.5 µM) on [Ca2+]c of an RPNEC. The results demonstrated in this figure were obtained from the same cell. (B) Summary of the extent of changes in fluorescence ratio (ΔF340/380) by various agonists and potassium-induced depolarization (KCl 60 mM). The number of tested cells are five to ten for each bar, respectively.

• 

  Fig. 2 ACh stimulates muscarinic receptors coupled to store Ca2+ release in RPNECs. (A) ACh induced increase in F340/380 is repeated in the presence and absence of extracellular Ca2+. (B) The ACh-induced ΔF340/380 is completely blocked by the application of atropine (1 µM), which is partially reversed by washout of atropine.

• 

  Fig. 3 M3 subtype of muscarinic receptors in RPNECs. (A) ACh (5 µM) was repetitively applied (closed bars) while measuring the F340/380 in RPNECs. The pretreatment with pF-HHSiD (0.3 µM) completely blocks the ACh-induced ΔF340/380. Note that the pretreatment with telenzepine has no effect on ACh-induced ΔF340/380. (B) McN-A-343 (1 µM), an M1-selective agonist, has no effect on [Ca2+]c. (C) The ACh-induced ΔF340/380 is not blocked by the pretreatment with himbacine (1 µM), an antagonist specific to M2 and M4 subtypes.

• 

  Fig. 4 RT-PCR analysis of M1, M2, M3, M4 and M5 subtypes in single RPNECs. Only the M3 signal was found in RPNECs. The leftmost column shows size markers in base pairs (bp).

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