P2X2 receptor expression by interstitial cells of Cajal in vas deferens implicated in semen emission
Introduction
Seminal release is divided into emission and ejaculation. Emission is the pulsatile transport of semen from storage in the epididymus through the lumen of the vas deferens to the prostatic urethra (Kolbeck and Steers, 1992). Neural stimulation of the vas deferens induces a biphasic contraction of smooth muscle leading to seminal emission (de Groat and Booth, 1980, Steers, 1994, Sneddon et al., 1996). Innervation of the vas deferens is derived from both the sympathetic and parasympathetic nervous systems. The majority of vas deferens neural input arises from sympathetic pre-ganglionic neurones in the lumbar spinal cord which synapse with post-ganglionic neurones in the pelvic accessory ganglion to innervate the vas deferens via the hypogastric nerve (Keast, 1992, Kolbeck and Steers, 1993, Kaleczyc et al., 1997). Parasympathetic pre-ganglionic cholinergic neurones originate from the lumbosacral spinal cord and traverse the major pelvic ganglion to innervate the vas deferens (Kolbeck and Steers, 1993, Kujat et al., 1993).
A small number of afferent fibres from the vas deferens travel through the hypogastric and pelvic nerves to the lumbosacral dorsal horn of the spinal cord (Kolbeck and Steers, 1992, Kolbeck and Steers, 1993). The role of afferents supplying the vas deferens is thought to relate to nociceptive or mechanoreceptive function (Floyd et al., 1976, Kolbeck and Steers, 1992).
The lumen of the vas deferens is surrounded by a columnar epithelial layer, a lamina propria, and three layers of smooth muscle fibres differentiated by orientation. This tissue is highly innervated, with muscle fibres adjacent to nerve varicosities (Burnstock, 1986). The majority of the post-ganglionic sympathetic fibres target the muscle layers, post-ganglionic parasympathetic (cholinergic) fibres target the lamina propria region and the inner longitudinal muscle layer. Peptidergic fibres target both regions (Keast, 1992). Sympathetic stimulation results in the co-release of noradrenaline and ATP (Vizi and Burnstock, 1988, Goncalves et al., 1995, Sneddon et al., 1996). Physiological experiments have shown that while noradrenaline mediates the secondary prolonged contraction phase, ATP is responsible for the initial rapid contraction of the vas deferens via P2X receptor activation (Friel, 1988, von Kügelgen and Starke, 1991, Burnstock, 1995, Sneddon et al., 1996, Nakanishi et al., 1997, Guitart et al., 1999, O’Connor et al., 1999).
P2X receptor subunits assemble to form homomeric or heteromeric ATP-gated ion channels (Brake et al., 1994, Valera et al., 1994, Lewis et al., 1995, Lê et al., 1998). The P2X1 receptor (P2X1R) was originally identified with the expression cloning of P2X1R cDNA from rat vas deferens (Valera et al., 1994). Northern blot analysis localized P2X1R mRNA expression within smooth muscle, neurones of the spinal cord and neonatal brain (Collo et al., 1996). Immunohistochemical analysis localized the presence of P2X1R expression to the outer longitudinal and circular muscle layers of vas deferens (Vulchanova et al., 1996, Worthington et al., 1999, Mulryan et al., 2000). Knock-out of the P2X1R subunit in mice significantly reduced sympathetic nerve-induced contraction of vas deferens, and induced male sterility by reducing the sperm count in the ejaculate, suggesting application in male contraception (Mulryan et al., 2000). Expression of the mRNA of P2X2R and P2X4R receptors subunits has also been detected by Northern blotting and reverse transcription polymerase chain reaction in vas deferens (Brake et al., 1994, Bo et al., 1995, Housley et al., 1995).
Here we report the localization of P2X2R mRNA and protein expression principally within the lamina propria region. We provide immunohistochemical evidence that the cells expressing the P2X2R subunit are interstitial cells of Cajal. The potential physiological significance of these findings is considered.
Section snippets
Materials and methods
Vas deferens tissue obtained from adult male Wistar strain rats, Swiss CD-1 strain mice and English short-hair guinea-pigs killed using pentobarbitone (Nembutal, 120 mg/kg, Virbac). Procedures followed guidelines approved by University of Auckland, Animal Ethics Committee.
Molecular characterization of P2X2 receptor mRNA isoforms within rat vas deferens
Two P2X2R subunit mRNA isoforms were isolated from rat vas deferens tissue. RT-PCR using mRNA from vas deferens and P2X2R specific primers yielded two distinct PCR products which migrated at approximately 700 and 480 bp with agarose gel electrophoresis (Fig. 1). Sequencing of the cloned PCR products indicated that these cDNAs were the P2X2–1R isoform described in PC12 cells (GenBank accession No. U14414) (Brake et al., 1994) and the P2X2–2R isoform which lacks 207 bp in the C-terminal coding
Discussion
Interstitial cells of Cajal (ICC) were resolved as a dense clustering of small cells with extensive reticular processes within the submuscosal (lamina propria) region of rat, mouse and guinea-pig vas deferens by neuron-specific enolase and c-Kit immunolabelling. This cell type expressed very high levels of the P2X2R subunit in all three species of animals examined. These P2XR subunits assemble in the cell membrane to form non-selective cation channels gated by extracellular ATP. The study
Acknowledgements
Supported by the Auckland Medical Research Foundation, the Marsden Fund and the New Zealand Lottery Grants Board.
References (66)
- et al.
A P2x purinoceptor cDNA conferring a novel pharmacological profile
FEBS Lett.
(1995) - et al.
Desensitization of the P2X2 receptor controlled by alternative splicing
FEBS Lett.
(1997) - et al.
Gene expression of P2X-receptors in the developing inner ear of the rat
Neurosci. Lett.
(1999) - et al.
Identification of a short form of the P2xR1-purinoceptor subunit produced by alternative splicing in the pituitary and cochlea
Biochem. Biophys. Res. Commun.
(1995) - et al.
An immunohistochemical study of interstitial cells of Cajal (ICC) in the equine gastrointestinal tract
Res. Vet. Sci.
(1999) Location and peptide content of pelvic neurons supplying the muscle and lamina propria of the rat vas deferens
J. Auton. Nerv. Syst.
(1992)- et al.
Origin of neurons supplying the vas deferens of the rat
J. Urol.
(1993) - et al.
Cholesterol feeding modulates spatial expression of TGF-beta 1 and beta 2 in aortas of Watanabe rabbits
Cytokine
(1995) A case for interstitial cells of Cajal as pacemakers and mediators of neurotransmission in the gastrointestinal tract
Gastroenterology
(1996)- et al.
Purinergic cotransmission: sympathetic nerves
Semin. Neurosci.
(1996)
Immunohistochemical distribution of c-Kit-positive cells and nitric oxide synthase-positive nerves in the guinea-pig small intestine
J. Auton. Nerv. Syst.
Blockade of kit signaling induces transdifferentiation of interstitial cells of Cajal to a smooth muscle phenotype
Gastroenterology
Origin of ATP release in the rat vas deferens: concomitant measurement of [3H]noradrenaline and [14C]ATP
Eur. J. Pharmacol.
Noradrenaline–ATP co-transmission in the sympathetic nervous system
Trends Pharmacol. Sci.
Development of interstitial cells of Cajal and pacemaking in mice lacking enteric nerves
Gastroenterology
New structural motif for ligand-gated ion channels defined by an ionotropic ATP receptor
Nature
Studies on the neurophysiology of the vas deferens
Invest. Urol.
Autonomic neuromuscular junctions: current developments and future directions
J. Anat.
Noradrenaline and ATP: cotransmitters and neuromodulators
J. Physiol. Pharmacol.
Cloning OF P2X5 and P2X6 receptors and the distribution and properties of an extended family of ATP-gated ion channels
J. Neurosci.
Physiology of male sexual function
Ann. Intern. Med.
Mechanosensitive afferent units in the hypogastric nerve of the cat
J. Physiol. (Lond.)
An ATP-sensitive conductance in single smooth muscle cells from the rat vas deferens
J. Physiol. (Lond.)
P2 receptor excitation of rodent hypoglossal motoneuron activity in vitro and in vivo: a molecular physiological analysis
J. Neurosci.
The arrangement and identification of axons innervating the vas deferens of the guinea-pig
J. Anat.
The c-Kit receptor, stem cell factor, and mast cells
Am. J. Pathol.
Comparison of corelease of noradrenaline and ATP evoked by hypogastric nerve stimulation and field stimulation in guinea-pig vas deferens
Naunyn-Schmiedeberg’s Arch. Pharmacol.
Expression of the P2X2 receptor subunit of the ATP-gated ion channel in the retina
Neuroreport
Adrenergic and purinergic components in bisected vas deferens from spontaneously hypertensive rats
Br. J. Pharmacol.
Role of the interstitial cells of Cajal in the control of gut motility
Br. J. Surg.
The expression of c-kit protein during oogenesis and early embryonic development
Biol. Reprod.
Cellular and molecular basis for electrical rhythmicity in gastrointestinal muscles
Annu. Rev. Physiol.
Expression of the P2X2 receptor subunit of the ATP-gated ion channel in the cochlea: implications for sound transduction and auditory neurotransmission
J. Neurosci.
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