Bhupal P. Bhetwal

Ca2+ sensitization pathways accessed by cholinergic neurotransmission in the murine gastric fundus

•  In smooth muscles, the sensitivity of contraction to Ca2+ can be increased by the phosphorylation of CPI‐17 and MYPT1, resulting in the inhibition of myosin light chain phosphatase (MLCP). •  Ca2+ sensitization of smooth muscle contraction has typically been studied by immersing muscles in solutions containing contractile agonists. •  However, stimulating muscles by bath‐applied agonists may not be equivalent to neurotransmitter release because different post‐junctional receptors may be activated in response to these different modes of stimulation. •  In this study we found that a bath‐applied cholinergic agonist activates Ca2+ sensitization mechanisms in gastric fundus smooth muscles that are different than those of cholinergic neurotransmission. Electrical field stimulation (EFS) only increased CPI‐17 phosphorylation, while bath‐applied carbachol increased both CPI‐17 and MYPT1 phosphorylation. •  With the cholinesterase inhibitor neostigmine present, both CPI‐17 and MYPT1 phosphorylation were increased by EFS. •  In fundus muscles of W/Wv mice which lack intramuscular interstitial cells of Cajal (ICC‐IMs), EFS alone increased both CPI‐17 and MYPT1 phosphorylation. •  These findings indicate that ACh availability determines which Ca2+ sensitization mechanisms are activated, and ICC‐IMs regulate the access of ACh to smooth muscles.

Spata6 is required for normal assembly of the sperm connecting piece and tight head–tail conjunction

Significance Male infertility due to acephalic spermatozoa has been reported in both animals and humans, but its cause remains largely unknown. Here we report that inactivation of Spata6, an evolutionarily conserved gene, in mice leads to failure in development of the connecting piece during late spermiogenesis, along with production of headless spermatozoa in the epididymis and ejaculates. The defects may be ascribed to the disrupted myosin-based microfilament transport mediated by SPATA6 through its interactions with myosin light-chain and heavy-chain subunits. This study not only unveils the process of sperm neck formation at both the ultrastructural and molecular levels, but also provides a genetic basis for the production of acephalic spermatozoa in both humans and animals. “Pinhead sperm,” or “acephalic sperm,” a type of human teratozoospermia, refers to the condition in which ejaculate contains mostly sperm flagella without heads. Family clustering and homogeneity of this syndrome suggests a genetic basis, but the causative genes remain largely unknown. Here we report that Spata6, an evolutionarily conserved testis-specific gene, encodes a protein required for formation of the segmented columns and the capitulum, two major structures of the sperm connecting piece essential for linking the developing flagellum to the head during late spermiogenesis. Inactivation of Spata6 in mice leads to acephalic spermatozoa and male sterility. Our proteomic analyses reveal that SPATA6 is involved in myosin-based microfilament transport through interaction with myosin subunits (e.g., MYL6).

Regulation of basal LC20 phosphorylation by MYPT1 and CPI-17 in murine gastric antrum, gastric fundus, and proximal colon smooth muscles

Background  Myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP) govern myosin light chain (LC20) phosphorylation and smooth muscle contraction. Rho kinase (ROK) inhibits MLCP, resulting in greater LC20 phosphorylation and force generation at a given [Ca2+]i. Here, we investigate the role of ROK in regulating LC20 phosphorylation and spontaneous contractions of gastric fundus, gastric antrum, and proximal colon smooth muscles.

RAN-binding protein 9 is involved in alternative splicing and is critical for male germ cell development and male fertility.

As a member of the large Ran-binding protein family, Ran-binding protein 9 (RANBP9) has been suggested to play a critical role in diverse cellular functions in somatic cell lineages in vitro, and this is further supported by the neonatal lethality phenotype in Ranbp9 global knockout mice. However, the exact molecular actions of RANBP9 remain largely unknown. By inactivation of Ranbp9 specifically in testicular somatic and spermatogenic cells, we discovered that Ranbp9 was dispensable for Sertoli cell development and functions, but critical for male germ cell development and male fertility. RIP-Seq and proteomic analyses revealed that RANBP9 was associated with multiple key splicing factors and directly targeted >2,300 mRNAs in spermatocytes and round spermatids. Many of the RANBP9 target and non-target mRNAs either displayed aberrant splicing patterns or were dysregulated in the absence of Ranbp9. Our data uncovered a novel role of Ranbp9 in regulating alternative splicing in spermatogenic cells, which is critical for normal spermatogenesis and male fertility.

Tra2β Protein Is Required for Tissue-specific Splicing of a Smooth Muscle Myosin Phosphatase Targeting Subunit Alternative Exon

Background: Alternative splicing of MYPT1 E23 defines fast versus slow smooth muscle. Results: Tra2β is required for the splicing of Mypt1 E23 in fast smooth muscle. Conclusion: Tra2β splicing factor confers unique contractile properties to fast smooth muscle. Significance: This is the first identification of a gene regulatory pathway conferring sensitivity to cGMP signaling in smooth muscle. Alternative splicing of the smooth muscle myosin phosphatase targeting subunit (Mypt1) exon 23 (E23) is tissue-specific and developmentally regulated and, thus, an attractive model for the study of smooth muscle phenotypic specification. We have proposed that Tra2β functions as a tissue-specific activator of Mypt1 E23 splicing on the basis of concordant expression patterns and Tra2β activation of Mypt1 E23 mini-gene splicing in vitro. In this study we examined the relationship between Tra2β and Mypt1 E23 splicing in vivo in the mouse. Tra2β was 2- to 5-fold more abundant in phasic smooth muscle tissues, such as the portal vein, small intestine, and small mesenteric artery, in which Mypt1 E23 is predominately included as compared with the tonic smooth muscle tissues, such as the aorta and inferior vena cava, in which Mypt1 E23 is predominately skipped. Tra2β was up-regulated in the small intestine postnatally, concordant with a switch to Mypt1 E23 splicing. Targeting of Tra2β in smooth muscle cells using SM22α-Cre caused a substantial reduction in Mypt1 E23 inclusion specifically in the intestinal smooth muscle of heterozygotes, indicating sensitivity to Tra2β gene dosage. The switch to the Mypt1 E23 skipped isoform coding for the C-terminal leucine zipper motif caused increased sensitivity of the muscle to the relaxant effects of 8-Br-cyclic guanosine monophosphate (cGMP). We conclude that Tra2β is necessary for the tissue-specific splicing of Mypt1 E23 in the phasic intestinal smooth muscle. Tra2β, by regulating the splicing of Mypt1 E23, sets the sensitivity of smooth muscle to cGMP-mediated relaxation.

Stk31 is Dispensable for Embryonic Development and Spermatogenesis in Mice

Mol. Reprod. Dev. 80: 786, 2013.

A novel class of somatic small RNAs similar to germ cell pachytene PIWI-interacting small RNAs

Background: Germ cells exclusively express PIWI-interacting small RNAs for transposon and gene regulation. Results: Somatic cells express similar RNAs that do not require known small RNA proteins and that partially complement mRNAs. Conclusion: These somatic small RNAs represent a novel small RNA population, which potentially regulates mRNA translation. Significance: Defining novel small RNAs is essential for elucidating the mechanisms that control gene expression. PIWI-interacting RNAs (piRNAs) are small noncoding RNAs that bind PIWI family proteins exclusively expressed in the germ cells of mammalian gonads. MIWI2-associated piRNAs are essential for silencing transposons during primordial germ cell development, and MIWI-bound piRNAs are required for normal spermatogenesis during adulthood in mice. Although piRNAs have long been regarded as germ cell-specific, increasing lines of evidence suggest that somatic cells also express piRNA-like RNAs (pilRNAs). Here, we report the detection of abundant pilRNAs in somatic cells, which are similar to MIWI-associated piRNAs mainly expressed in pachytene spermatocytes and round spermatids in the testis. Based on small RNA deep sequencing and quantitative PCR analyses, pilRNA expression is dynamic and displays tissue specificity. Although pilRNAs are similar to pachytene piRNAs in both size and genomic origins, they have a distinct ping-pong signature. Furthermore, pilRNA biogenesis appears to utilize a yet to be identified pathway, which is different from all currently known small RNA biogenetic pathways. In addition, pilRNAs appear to preferentially target the 3′-UTRs of mRNAs in a partially complementary manner. Our data suggest that pilRNAs, as an integral component of the small RNA transcriptome in somatic cell lineages, represent a distinct population of small RNAs that may have functions similar to germ cell piRNAs.

Role of Telokin in Regulating Murine Gastric Fundus Smooth Muscle Tension

Telokin phosphorylation by cyclic GMP-dependent protein kinase facilitates smooth muscle relaxation. In this study we examined the relaxation of gastric fundus smooth muscles from basal tone, or pre-contracted with KCl or carbachol (CCh), and the phosphorylation of telokin S13, myosin light chain (MLC) S19, MYPT1 T853, T696, and CPI-17 T38 in response to 8-Bromo-cGMP, the NO donor sodium nitroprusside (SNP), or nitrergic neurotransmission. We compared MLC phosphorylation and the contraction and relaxation responses of gastric fundus smooth muscles from telokin-/- mice and their wild-type littermates to KCl or CCh, and 8-Bromo-cGMP, SNP, or nitrergic neurotransmission, respectively. We compared the relaxation responses and telokin phosphorylation of gastric fundus smooth muscles from wild-type mice and W/W V mice which lack ICC-IM, to 8-Bromo-cGMP, SNP, or nitrergic neurotransmission. We found that telokin S13 is basally phosphorylated and that 8-Bromo-cGMP and SNP increased basal telokin phosphorylation. In muscles pre-contracted with KCl or CCh, 8-Bromo-cGMP and SNP had no effect on CPI-17 or MYPT1 phosphorylation, but increased telokin phosphorylation and reduced MLC phosphorylation. In telokin-/- gastric fundus smooth muscles, basal tone and constitutive MLC S19 phosphorylation were increased. Pre-contracted telokin-/- gastric fundus smooth muscles have increased contractile responses to KCl, CCh, or cholinergic neurotransmission and reduced relaxation to 8-Bromo-cGMP, SNP, and nitrergic neurotransmission. However, basal telokin phosphorylation was not increased when muscles were stimulated with lower concentrations of SNP or when the muscles were stimulated by nitrergic neurotransmission. SNP, but not nitrergic neurotransmission, increased telokin Ser13 phosphorylation in both wild-type and W/W V gastric fundus smooth muscles. Our findings indicate that telokin may play a role in attenuating constitutive MLC phosphorylation and provide an additional mechanism to augment gastric fundus mechanical responses to inhibitory neurotransmission.

Premature contractions of the bladder are suppressed by interactions between TRPV4 and SK3 channels in murine detrusor PDGFRα cells.

During filling, urinary bladder volume increases dramatically with little change in pressure. This is accomplished by suppressing contractions of the detrusor muscle that lines the bladder wall. Mechanisms responsible for regulating detrusor contraction during filling are poorly understood. Here we describe a novel pathway to stabilize detrusor excitability involving platelet-derived growth factor receptor-α positive (PDGFRα+) interstitial cells. PDGFRα+ cells express small conductance Ca2+-activated K+ (SK) and TRPV4 channels. We found that Ca2+ entry through mechanosensitive TRPV4 channels during bladder filling stabilizes detrusor excitability. GSK1016790A (GSK), a TRPV4 channel agonist, activated a non-selective cation conductance that coupled to activation of SK channels. GSK induced hyperpolarization of PDGFRα+ cells and decreased detrusor contractions. Contractions were also inhibited by activation of SK channels. Blockers of TRPV4 or SK channels inhibited currents activated by GSK and increased detrusor contractions. TRPV4 and SK channel blockers also increased contractions of intact bladders during filling. Similar enhancement of contractions occurred in bladders of Trpv4−/− mice during filling. An SK channel activator (SKA-31) decreased contractions during filling, and rescued the overactivity of Trpv4−/− bladders. Our findings demonstrate how Ca2+ influx through TRPV4 channels can activate SK channels in PDGFRα+ cells and prevent bladder overactivity during filling.

Reply from Kenton M. Sanders, Bhupal P. Bhetwal and Brian A. Perrino

Contractile tone is a requisite factor in normal filling and emptying of the proximal stomach. Neural inputs are key regulators of tone (thus gastric volume and compliance). During eating there is net inhibitory drive from nitrergic/purinergic neurons, and as the proximal stomach delivers food to the distal stomach, cholinergic influences gradually increase tone, empty the fundus and restore the resting volume of the stomach. For many years investigators have attempted to determine what cells and mechanisms mediate the post-junctional responses to neurotransmitters in the fundus. Knowing the answers to these questions might reveal useful therapeutics for conditions, such as dyspepsia and gastroparesis, that affect many patients worldwide. Our recent study (Bhetwal et al. 2013) was designed to determine whether the same pathways are activated by neurotransmitters released from enteric motor neurons as those activated by neurotransmitter substances added to a solution bathing a muscle strip. We reasoned that a relatively tiny mass of neurotransmitter released in a punctate manner from nerve varicosities might achieve different concentration profiles and bind to different populations of receptors compared to high-mass amounts of bath-applied transmitter diffusing freely through the extracellular spaces in muscles. Our findings indicate that the post-junctional receptors activated during cholinergic neurotransmission must be different from those activated when muscles are bathed in cholinergic agonists. Use of W/WV mice allowed us to further refine the role of interstitial cells of Cajal (ICC) in cholinergic neurotransmission, and we do not believe our previous findings were contradicted by the new data, as Professor Goyal suggests. We found that when ACh is released from neurons, there is an increase in the phosphorylation of CPI-17, a protein known to increase Ca2+ sensitivity in smooth muscle contraction. We did not resolve phosphorylation of MYPT1, another mediator of enhanced Ca2+ sensitivity, when ACh was released from neurons, but MYPT1 was clearly phosphorylated when carbachol was added to bath solutions. Thus, our conclusion that neurally released ACh binds mainly to receptors on cells other than smooth muscle cells (SMCs) is based on these data. Phosphorylation of CPI-17 appeared to result from a Ca2+-dependent protein kinase C. Then we attempted to perturb the system by inhibiting the metabolism of ACh released from neurons or by using W/WV muscles missing most of the ICC-IM that form close associations with the terminals of motor neurons. In both conditions ACh released from neurons caused phosphorylation of MYPT1 as a post-junctional mechanism activated by cholinergic nerve stimulation. An explanation for these data is that ACh released from motor neurons binds primarily to receptors on ICC, and this results in depolarization of these cells by activation of Ca2+-activated Cl− channels. Depolarization of ICC conducts to SMCs via gap junctions that exist between ICC and SMCs in the stomach (Komuro et al. 1999; Horiguchi et al. 2003). Depolarization of SMCs enhances Ca2+ entry via voltage-dependent Ca2+ channels. Removing ICC may increase the access of the neurotransmitter to receptors on SMCs that are coupled through G proteins to Rho kinase and phosphorylation of MYPT1. Removing the tiny volumes formed between motor nerve terminals and ICC may reduce the rate of metabolism of ACh because post-junctional concentrations of neurotransmitter may not reach levels as high when released generally into the interstitium. Therefore, the ‘barrier’ against overflow of ACh onto SMC receptors produced by junctions of ICC and enteric nerve terminals does not physically restrict the diffusion of neurotransmitter but rather the amount of available transmitter is reduced by the robust enzymatic activity of acetylcholine esterase. Professor Goyal seems to favour the traditional view that neurotransmitters course freely through the interstitial volume, and responses in gastrointestinal (GI) muscles are the sole result of neurotransmitter binding to SMC receptors. However, morphology shows that the receptive field in GI muscles is populated by at least three types of electrically coupled cells (SMCs, ICC and another interstitial cell that is selectively labelled with antibodies against PDGFRα– PDGFRα+ cells), and we have referred to the receptive field in these muscles as the SIP syncytium (Sanders et al. 2012). When a neurotransmitter is released from a nerve varicosity the response is determined by multiple factors, as discussed in more depth elsewhere (Sanders et al. 2010): (i) the concentration profile of the transmitter as it diffuses within the interstitium, (ii) the expression of receptors with affinity for the neurotransmitter by cells near the site of release, (iii) efficacious coupling of receptors to effector mechanisms, and (iv) the rate of metabolism, uptake of the transmitter, or other means of reducing transmitter concentration. Since all SIP cells express receptors for enteric motor neurotransmitters, it is likely that the post-junctional response is highly integrated and not as simple as transduction by SMCs. Redundant receptors and pathways may provide a ‘safety factor’ when normal response mechanisms are lost or damaged. Our findings have shown ICC to be a first-line mediator of cholinergic electrophysiological responses (Ward et al. 2000), but our recent study (Bhetwal et al. 2013) demonstrates that contractile responses are preserved when ICC are reduced in numbers via recruitment of SMC Ca2+ sensitization pathways. Professor Goyal seems to suggest that if contractile responses to motor nerve stimulation are preserved in the absence of ICC, then these cells are irrelevant to GI motility. This view may be too simplistic, and of greater importance is the likelihood that the causes for GI motility disorders may not be as black and white as traditional concepts might imply. The question of whether cholinergic responses in animals lacking ICC are normal might be the most cogent point for future investigation. Loss of electrophysiological responsiveness and increasing the gain on Ca2+ sensitization mechanisms by recruiting MYPT1 phosphorylation, as occur in fundus muscles lacking ICC, may distort responses to other agonists, such as prostaglandins, hormones, or nitric oxide, and alter compliance responses during gastric filling and/or the rate at which the proximal stomach empties into the distal stomach. Abnormal responses to otherwise normal signalling by the many bioactive substances at play in the postprandial stomach may be at the heart of functional dyspepsia and adversely affect the rate of gastric emptying. We would suggest that normal responses are likely to result from the functional apparatus (e.g. the SIP syncytium) developed through evolution in wild-type animals. Since evolution is primarily concerned with function, and not so much with form, it is imprudent to expect that synaptic or post-junctional structures mediating motor responses in visceral smooth muscles must be identical to synaptic structures in the CNS or neuromuscular junctions in skeletal muscles. Therefore, it may be premature to discount ICC and label them as ‘expendable’ when as yet, we have an incomplete understanding of the mechanisms regulating gastric compliance and little or no certainty about the pathophysiological basis for gastric motility disorders.