Differential effects of soluble and particulate guanylyl cyclase on Ca(2+) sensitivity in airway smooth muscle.

Maximal relaxation of airway smooth muscle (ASM) in response to atrial natriuretic peptide (ANP), which stimulates particulate guanylyl cyclase (pGC), is less than that produced by nitric oxide (NO) and other compounds that stimulate soluble guanylyl cyclase (sGC). We hypothesized that stimulation of pGC relaxes ASM only by decreasing intracellular Ca(2+) concentration ([Ca(2+)](i)), whereas stimulation of sGC decreases both [Ca(2+)](i) and the force developed for a given [Ca(2+)](i) (i.e., the Ca(2+) sensitivity) during muscarinic stimulation. We measured the relationship between force and [Ca(2+)](i) (using fura 2) under control conditions (using diltiazem to change [Ca(2+)](i)) and during exposure to ANP, diethylamine-NO (DEA-NO), sodium nitroprusside (SNP), and the Sp diastereoisomer of beta-phenyl-1,N(2)-etheno-8-bromoguanosine-3',5'-cyclic monophosphorothionate (Sp-8-Br-PET-cGMPS), a cell-permeant analog of cGMP. Addition of DEA-NO, SNP, or Sp-8-Br-PET-cGMPS decreased both [Ca(2+)](i) and force, causing a significant rightward shift of the force-[Ca(2+)](i) relationship. In contrast, with ANP exposure, the force-[Ca(2+)](i) relationship was identical to control, such that ANP produced relaxation solely by decreasing [Ca(2+)](i). Thus, during muscarinic stimulation, stimulation of pGC relaxes ASM exclusively by decreasing [Ca(2+)](i), whereas stimulation of sGC decreases both [Ca(2+)](i) and Ca(2+) sensitivity.     

Identification of telocytes in the upper lamina propria of the human urinary tract

The upper lamina propria (ULP) area of interstitial cells (IC) has been studied extensively in bladder, but is rather unexplored in the rest of the urinary tract. This cell layer is intriguing because of the localization directly underneath the urothelium, the intercellular contacts and the close relationship with nerve endings and capillaries. In this study, we examine the ULP layer of IC in human renal pelvis, ureter and urethra, and we make a comparison with ULP IC in bladder. Tissue was obtained from normal areas in nephrectomy, cystectomy and prostatectomy specimens, and processed for morphology, immunohistochemistry and electron microscopy. A morphological and immunohistochemical phenotype for the ULP IC was assessed and region-dependent differences were looked for. The ULP IC in renal pelvis, ureter and urethra had a similar ultrastructural phenotype, which differed somehow from that of bladder IC, that is, thinner and longer cytoplasmic processes, no peripheral actin filaments and presence of dense core granules and microtubules. Together with their immunohistochemical profile, these features are most compatible with the phenotype of telocytes, a recently discovered group of stromal cells. Based on their global ultrastructural and immunohistochemical phenotype, ULP IC in human bladder should also be classified as telocytes. The most striking immunohistochemical finding was the variable expression of oestrogen receptor (ER) and progesterone receptor (PR). The functional relevance of ULP telocytes in the urinary tract remains to be elucidated, and ER and PR might therefore be promising pharmacological research targets.     

Nucleotidyl cyclase activity of soluble guanylyl cyclase in intact cells

Soluble guanylyl cyclase (sGC) is activated by nitric oxide (NO) and generates the second messenger cyclic GMP (cGMP). Recently, purified sGC α₁β₁ has been shown to additionally generate the cyclic pyrimidine nucleotides cCMP and cUMP. However, since cyclic pyrimidine nucleotide formation occurred only the presence of Mn²⁺ but not Mg²⁺, the physiological relevance of these in vitro findings remained unclear. Therefore, we studied cyclic nucleotide formation in intact cells. We observed NO-dependent cCMP- and cUMP formation in intact HEK293 cells overexpressing sGC α₁β₁ and in RFL-6 rat fibroblasts endogenously expressing sGC, using HPLC–tandem mass spectrometry. The identity of cCMP and cUMP was unambiguously confirmed by HPLC–time-of-flight mass spectrometry. Our data indicate that cCMP and cUMP play second messenger roles and that Mn²⁺ is a physiological sGC cofactor.     

Interaction of atrial natriuretic factor and endothelin-1 signals through receptor guanylate cyclase in pulmonary artery endothelial cells

The endothelial cell has a unique intrinsic feature: it produces a most potent vasopressor peptide hormone, endothelin (ET-1), yet it also contains a signaling system of an equally potent hypotensive hormone, atrial natriuretic factor (ANF). This raises two related curious questions: does the endothelial cell also contain an ET-1 signaling system? If yes, how do the two systems interact with each other? The present investigation was undertaken to determine such a possibility. Bovine pulmonary artery endothelial (BPAE) cells were chosen as a model system. Identity of the ANF receptor guanylate cyclase was probed with a specific polyclonal antibody to the 180 kDa membrane guanylate cyclase (mGC) ANF receptor. A Western-blot analysis of GTP-affinity-purified endothelial cell membrane proteins recognized a 180 kDa band; the same antibody inhibited the ANF-stimulated guanylate cyclase activity; the ANF-dependent rise of cyclic GMP in the intact cells was dose-dependent. By affinity cross-linking technique, a predominant 55 kDa membrane protein band was specifically labeled with [¹²⁵I]ET-1. ET-1 treatment of the cells showed a migration of the protein kinase C (PKC) activity from cytosol to the plasma membrane; ET-1 inhibited the ANF-dependent production of cyclic GMP in a dose-dependent fashion with an EC₅₀ of 100 nM. This inhibitory effect was duplicated by phorbol 12-myristate 13-acetate (PMA), a known PKC-activator. The EC₅₀ of PMA was 5 nM. A PKC inhibitor, 1-(5-isoquinolinyl-sulfonyl)-2-methyl piperazine (H-7), blocked the PMA-dependent attenuation of ANF-dependent cyclic GMP formation. These results demonstrate that the 180 kDa mGC-coupled ANF and ET-1 signaling systems coexist in endothelial cells and that the ET-1 signal negates the ANF-dependent guanylate cyclase activity and cyclic GMP formation. Furthermore, these results support the paracrine and/or autocrine role of ET-1.     

Serum response factor, its cofactors, and epithelial-mesenchymal signaling in urinary bladder smooth muscle formation

Little is known about the mechanism of bladder smooth muscle differentiation. We hypothesize that epithelial-mesenchymal signaling induces the expression of smooth muscle proteins in bladder mesenchyme resulting in smooth muscle differentiation. We confirmed that smooth muscle differentiation in the mouse urinary bladder occurs first at gestational day 14 (E14) based upon immunohistochemical localization of smooth muscle alpha-actin (SMAA). To investigate murine bladder smooth muscle differentiation and epithlelial-mesenchymal signaling in the developing bladder, we analyzed gene expression profiles of intact embryonic murine bladders and separated epithelial and mesenchymal components at embryonic days E13, E14, E15, E16, and postnatal day 1 (P1). Using cDNA microarray, we identified regulators of vascular smooth muscle differentiation in bladder mesenchyme, including serum response factor (SRF) and its cofactors, ELK1 and SRF accessory protein (SAP)1, as well as two SRF-associated pathways, angiotension receptor II and transforming growth factor- beta2. Immunohistochemistry showed diffuse expression of SRF in the bladder at E12 with localization of expression to the peripheral mesenchyme at E13 and E14. Our results suggest that bladder smooth muscle differentiation may share a similar gene expression program as occurs during vascular smooth muscle differentiation. The unique structure of the urinary bladder makes it an ideal model for studies of smooth muscle differentiation and epithelial-mesenchymal signaling.     

Contraction kinetics and myosin isoform composition in smooth muscle from hypertrophied rat urinary bladder

Mechanical properties and isoform composition of myosin heavy and light chains were studied in hypertrophying rat urinary bladders. Growth of the bladder was induced by partial ligation of the urethra. Preparations were obtained after 10 days. In maximally activated skinned preparations from the hypertrophying tissue, the maximal shortening velocity and the rate of force development following photolytic release of ATP were reduced by about 20 and 25%, respectively. Stiffness was unchanged. The relative content of the basic isoform of the essential 17 kDa myosin light chain was doubled in the hypertrophied tissue. The expression of myosin heavy chain with a 7 amino acid insert at the 25K/50K region was determined using a peptide-derived antibody against the insert sequence. The relative amount of heavy chain with insert was decreased to 50%, in the hypertrophic tissue. The kinetics of the cross-bridge turn-over in the newly formed myosin in the hypertrophic smooth muscle is reduced, which might be related to altered expression of myosin heavy or light chain isoforms. © 1996 Wiley-Liss, Inc.     

Peptides from the N-terminus of the atrial natriuretic factor prohormone enhance guanylate cyclase activity and increase cyclic GMP levels in a wide variety of tissues

The 98 amino acid (a. a.) N-terminus of the 126 a. a. atrial natriuretic factor (ANF) prohormone contains three peptides consisting of a. a. 1–30 (proANF 1–30), a. a. 31–67 (proANF 31–67) and a. a. 79–98 (proANF 79–98) with blood pressure lowering, sodium and/or potassium excreting properties similar to atrial natriuretic factor (a. a. 99–126, C-terminus of prohormone). ProANF 1–30 and proANF 31–67 have separate and distinct receptors from ANF in both vasculature and in the kidney to help mediate the above effects. At the cellular level proANFs 1–30, 31–67, and 79–98 as well as ANF's effects are mediated by enhancement of the guanylate cyclase (EC 4.6.1.2) — cyclic GMP system in vasculature and in the kidney. These peptides from the N-terminus of the ANF prohormone circulate normally in man and in all animal species tested. The object of the present investigation was to determine if these peptides have the ability to enhance either guanylate cyclase and/or adenylate cyclase in a variety of other tissues in addition to kidney and vasculature. ProANF 1–30, proANF 31–67, proANF 79–98, and ANF all increased rat lung, liver, heart and testes, but not spleen, particulate guanylate cyclase 2- to 3-fold at their 100 nM concentrations. Dose response curves revealed that maximal stimulation of particulate guanylate cyclase activity by these newly discovered peptides was at their 1 M concentrations, with no further increase in activity above their 1 M concentrations. Half-maximal (EC₅₀) enhancement of particulate guanylate cyclase occurred at 0.15 ± 0.01, 0.3 ± 0.02, 0.5 ± 0.03, and 0.9 ± 0.03 nM for proANF 1–30, proANF 31–67, proANF 79–98 and ANF, respectively. ProANFs 1–30, 31–67, 79–98, and 99–126 (i.e., ANF) each increased cyclic GMP but not cyclic AMP levels in tissue slices of liver, lung, small intestine, heart, and testes. None of these peptides enhanced either adenylate cyclase or the soluble 100,000 G form of guanylate cyclase. The ability of these N-terminal peptides to enhance particulate guanylate cyclase activity in a wide variety of tissues suggests that they may have effects in a much wider variety of tissues than presently thought.     

Differences in the proportion of collagen and muscle in the canine lower urinary tract with regard to gonadal status and gender

Gonadectomy not only affects hormonal homeostasis but also alters the turnover of different components of the extracellular matrix in urogenital tissues. Collagen is an important component of the bladder and urethral walls and thus crucial for the mechanical properties of normal lower urinary tract (LUT) functions. This study aimed at investigating the possibility of differences in the proportion of collagen and muscle tissues in the LUT of intact and gonadectomised male and female dogs. Twenty clinically healthy dogs were used including 10 sexually intact dogs (5 males, 5 anoestrus females) and 10 gonadectomised dogs (4 males and 6 females). Four regions of the LUT, i.e. body and neck of the bladder as well as proximal and distal urethra were collected. The tissue sections were stained with Masson's Trichrome. Quantitative evaluation of the blue-stained area for collagen and red-counterstained area for muscle was performed using colour image analysis. The relative proportion of collagen and muscle significantly differed with the gonadal status, the gender and the region. Overall, gonadectomised dogs had a higher (P < 0.001) proportion of collagen and consequently a lower (P < 0.001) proportion of muscle than intact dogs. Regardless of gonadal statuses, females had a higher (P < 0.05) proportion of collagen and a lower (P < 0.05) proportion of muscle tissues than males. Gender differences were found in all four regions of the LUT in intact dogs but only in proximal urethra in gonadectomised dogs where spayed females had a higher (P < 0.05) proportion of collagen and less muscle (P < 0.05). Regional differences were observed in females; a higher proportion of collagen and therefore less muscle were found in the urethra compared with the bladder. Proportional differences in collagen and muscle between intact and gonadectomised animals suggest a relation of different hormonal statuses to structural changes in the canine LUT. Excessive collagen deposits and less muscular volume may impair structural and functional integrity of the LUT which may associate with the development of post-neutering urinary incontinence in the dog.     

Big-conductance Ca2+-activated K+ channels in physiological and pathophysiological urinary bladder smooth muscle cells

Contraction and relaxation of urinary bladder smooth muscle cells (UBSMCs) represent the important physiological functions of the bladder. Contractile responses in UBSMCs are regulated by a number of ion channels including big-conductance Ca²⁺- activated K⁺ (BK) channels. Great progress has been made in studies of BK channels in UBSMCs. The intent of this review is to summarize recent exciting findings with respect to the functional interactions of BK channels with muscarinic receptors, ryanodine receptors (RyRs) and inositol triphosphate receptors (IP₃Rs) as well as their functional importance under normal and pathophysiological conditions. BK channels are highly expressed in UBSMCs. Activation of muscarinic M₃ receptors inhibits the BK channel activity, facilitates opening of voltage-dependent Ca²⁺ (Ca_V) channels, and thereby enhances excitability and contractility of UBSMCs. Signaling molecules and regulatory mechanisms involving RyRs and IP₃Rs have a significant effect on functions of BK channels and thereby regulate cellular responses in UBSMCs under normal and pathophysiological conditions including overactive bladders. Moreover, BK channels may represent a novel target for the treatment of bladder dysfunctions.