Resting membrane potentials of pacemaker and non pacemaker areas in rat uterus

Microelectrode studies of pacemaker and non pacemaker cells in pregnant rat uterus have shown the pacemaker areas to have a constant value of RMP throughout pregnancy which was always significantly smaller than that of non pacemaker cells. The development of new pacemaker areas was associated with membrane depolarization. A number of agents and conditions which caused membrane depolarization also induced pacemaker activity in previously non pacemaker areas but did so at different levels of membrane depolarization. Potassium depolarization failed to induce pacemaker activity. It is concluded that a low level of RMP is an important factor but not sufficient alone to explain the occurence of pacemaker activity.The resting membrane potentials (RMP) of spontaneously active smooth muscles are appreciably smaller than those of nonspontaneously active muscles (3) and comparable in magnitude to those of other tissues showing spontaneous activity such as the frog sinus venosus(7), rabbit sino-auricular node (10) and embryonic heart muscle (6). In intestinal smooth muscle, where all cells appear to be spontaneously active, a clear relationship can be demonstrated between fluctuating levels of RMP and the incidence of action potential activity, and the ionic and metabolic basis of slow wave activity has been extensively investigated (5, 8). In other smooth muscles, such as the ureter and uterus, where electrical activity arises from pacemaker areas (11, 12), the underlying causes of spontaneous activity are less well understood and the relationships between pacemaker activity, RMP and excitability have not been clearly defined. As an initial approach to studying this problem we have chosen to investigate the relationship between RMP and pacemaker activity in the uterus of the pregnant rat.     

Development and differentiation of the ureteric bud into the ureter in the absence of a kidney collecting system

Six1-/- mice were found to have apparently normal ureters in the absence of a kidney, suggesting that the growth and development of the unbranched ureter is largely independent of the more proximal portions of the UB which differentiates into the highly branched renal collecting system. Culture of isolated urinary tracts (from normal and mutant mice) on Transwell filters was employed to study the morphogenesis of this portion of the urogenital system. Examination of the ureters revealed the presence of a multi-cell layered tubule with a lumen lined by cells expressing uroplakin (a protein exclusively expressed in the epithelium of the lower urinary tract). Cultured ureters of both the wild-type and Six1 mutant become contractile and undergo peristalsis, an activity preceded by the expression of alpha-smooth muscle actin (alphaSMA). Treatment with a number of inhibitors of signaling molecules revealed that inhibition of PI3 kinase dissociates the developmental expression of alphaSMA from ureter growth and elongation. Epidermal growth factor also perturbed smooth muscle differentiation in culture. Moreover, the peristalsis of the ureter in the absence of the kidney in the Six1-/- mouse indicates that the development of this clinically important function of ureter (peristaltic movement of urine) is not dependent on fluid flow through the ureter. In keeping with this, isolated ureters cultured in the absence of surrounding tissues elongate, differentiate and undergo peristalsis when cultured on a filter and undergo branching morphogenesis when cultured in 3-dimensional extracellular matrix gels in the presence of a conditioned medium derived from a metanephric mesenchyme (MM) cell line. In addition, ureters of Six1-/- urinary tracts (i.e., lacking a kidney) displayed budding structures from their proximal ends when cultured in the presence of GDNF and FGFs reminiscent of UB budding from the wolffian duct. Taken together with the above data, this indicates that, although the distal ureter (at least early in its development) retains some of the characteristics of the more proximal UB, the growth and differentiation (i.e., development of smooth muscle actin, peristalsis and uroplakin expression) of the distal non-branching ureter are inherent properties of this portion of the UB, occurring independently of detectable influences of either the undifferentiated MM (unlike the upper portion of the ureteric bud) or more differentiated metanephric kidney. Thus, the developing distal ureter appears to be a unique anatomical structure which should no longer be considered as simply the non-branching portion of the ureteric bud. In future studies, the ability to independently analyze and study the portion of the UB that becomes the renal collecting system and that which becomes the ureter should facilitate distinguishing the developmental nephrome (renal ontogenome) from the ureterome.     

Characterization of spontaneous electrical activity of the urinary tract: Ureter,bladder, urethra

The study aimed to characterize spontaneous electrical activity of the ureter, urinary bladder and urethra as well as their interrelationship. The basic parameters of pacemaker activity (amplitude, frequency, peak rise rate, peak rise time, peak half-width) were comparatively analyzed in each of the active areas. Out of the three areas compared, the ureteral rhythmogenic zone displayed the maximum amplitude and apex formation rate. Under conditions of urine influx from the ureter into the bladder and isolation of these organs from the urethra, the amplitude and peak rise rate in the latter decreased by almost 20%. At the same time, all the parameters of the ureter and bladder remained intact. Complete block of urine influx into the bladder by transecting the ureter at the appropriate area led to a slight decrease in the amplitude of action potentials, peak rise rate and rhythmogenicity frequency in the bladder, respectively, by 14.2, 12.5 and 16% at the constancy of other parameters of its activity. Subsequent isolation of the bladder from the urethra had no appreciable effect on the altered parameters of the former. The similar tendency towards a reduction of the parameters was observed under the same conditions in the urethra. Thus, a relationship was revealed between autonomous activities of the ureter, bladder and urethra. The regulatory role in this process is provided by the urine flow through these organs.     

Abnormalities in ureter and kidney development in mice given acetazolamide-amiloride or dimethadione (DMO) during embryogenesis.

These experiments more accurately define the effects of the combination acetazolamide-amiloride or a single dose of dimethadione (DMO), the active metabolite of trimethadione, on the development of the ureter. When acetazolamide-amiloride was administered in C57BL/6NCrlBR mice on day 9, 9.5, or 10 of gestation (plug = day 0) a second ureter was formed, anterior to the original ureter, inducing a second kidney. The second ureter then fails to make a connection with the developing bladder and remains attached to the mesonephric duct. The mesonephric duct becomes the vas deferens in the male and deteriorates completely in the female leading to either a restricted ureter or a blocked ureter depending on the sex of the fetus. Administration of a single dose of DMO between gestational day 9 and 10.3 produced both renal agenesis and ureters of varying lengths. Some ureters were of normal length with a tuft of one or two nephrons at their tip, while others were one half or one quarter of their normal length. In some instances the ureter was completely absent. The reason for this strong effect on the ureter is unknown.     

Cascade Bioassay Evidence for the Existence of Urothelium-Derived Inhibitory Factor in Guinea Pig Urinary Bladder

Our aim was to investigate whether guinea pig urothelium-derived bioactivities compatible with the existence of urothelium-derived inhibitory factor could be demonstrated by in vitro serial bioassay and whether purinergic P1 receptor agonists, nitric oxide, nitrite or prostaglandins might explain observed activities. In a cascade superfusion system, urothelium-denuded guinea pig ureters were used as bioassay tissues, recording their spontaneous rhythmic contractions in presence of scopolamine. Urothelium-intact or -denuded guinea pig urinary bladders were used as donor tissues, stimulated by intermittent application of carbachol before or during the nitric oxide synthase inhibitor N^G-nitro-L-arginine methyl ester (L-NAME), the adenosine/P1 nucleoside receptor antagonist 8-(p-sulfophenyl)theophylline (8-PST) or the cyclo-oxygenase inhibitor diclofenac infused to bath donor and bioassay tissues. The spontaneous contractions of bioassay ureters were unaltered by application of carbachol 1–5 µM in the presence of scopolamine 5–30 µM. When carbachol was applied over the urothelium-denuded bladder, the assay ureter contraction rate was unaltered. Introducing carbachol over the everted urothelium-intact bladder significantly inhibited the contraction frequency of the assay ureter, suggesting the transfer of an inhibitory activity from the bladder to the assay ureter. The transmissible inhibitory activity was not markedly antagonized by L-NAME, 8-PST or diclofenac, while L-NAME nearly abolished nitrite release from the urothelium-intact bladder preparations. We suggest that urothelium-derived inhibitory factor is a transmissible entity over a significant distance as demonstrated in this novel cascade superfusion assay and seems less likely to be nitric oxide, nitrite, an adenosine receptor agonist or subject to inhibition by administration of a cyclo-oxygenase inhibitor.     

Inhibitory influences of vagal afferences on the oesophageal EMG peristaltic pattern

The influence of vagal afferents on the EMG peristaltic pattern was studied in pigeon oesophagus. Bilateral vagotomy did not abolish the primary peristalsis, but induced significant modifications of the peristaltic pattern parameters. Vagal afferent stimulation induced an inhibitory effect consisting of a temporary break or definitive block of the EMG peristaltic activity already in progress. Vagal afferent stimulation also induced a reduction of the spontaneous EMG activity and this effect was abolished either by glossopharyngeal bilateral section or ganglionic block. Likewise vagal afferent stimulation, the crop distension caused inhibitory effects on EMG peristaltic pattern. This effect was abolished by bilateral vagotomy. These results indicate that vagal afferents, originating from the crop, could influence the central neurons responsible for the peristaltic motor programme.     

Interstitial cell of Cajal-like cells in the upper urinary tract

Autorhythmicity in the upper urinary tract (UUT) has long been considered to arise in specialized atypical smooth muscle cells (SMC) predominately situated in the most proximal regions of the pyeloureteric system. These atypical SMC pacemakers have been thought to trigger adjacent electrically-quiescent typical SMC to fire action potentials which allow an influx of Ca2+ and the generation of muscle contraction. More recently, the presence of cells with many of the morphological, electrical and immunohistochemical characteristics of interstitial cells of Cajal (ICC), the pacemaker cells of the gastrointestinal tract, have been located in many regions of both the upper and lower urinary tract. This article reviews the evidence from the literature and from our laboratory supporting a role of both atypical SMC and ICC-like cells in the initiation and propagation of pyeloureteric peristalsis in the UUT. We propose a new model in which there are 2 populations of pacemaker cells, high frequency atypical SMC and lower frequency ICC-like cells, both of which can drive electrically-quiescent typical SMC. The relative presence of these 2 populations of pacemaker cells and the relatively-long refractoriness of typical SMC determines the decreasing frequency of contraction with distance from the renal fornix. In the absence of the proximal pacemaker drive from atypical SMC after pyeloureteral/ureteral obstruction or surgery, ICC-like cell pacemaking provides a compensatory mechanism allowing the ureter to maintain rudimentary peristaltic waves and movement of urine from the pyelon towards the bladder.     

Flow of urine through the ureter: a collapsible, muscular tube undergoing peristalsis

In steady flow through nonuniform collapsible tubes a key concept is the compressive zone, at which flow limitation can occur at both high and low Reynolds numbers. Ureteral peristalsis can be considered as a series of compressive zones, corresponding to waves of active muscular contraction, that move at near-constant speed along the ureter towards the bladder. One-dimensional, lubrication-theory analysis shows that peristalsis can pump urine from kidney into the bladder only at relatively low mean rates of urine flow. Under these circumstances isolated boluses of urine are propelled steadily through the ureter (assumed uniform) by the contraction waves. At higher mean rates of flow the behavior depends on whether the frequency of peristalsis is higher or lower than a critical value. For frequencies above the critical value steady propagation of boluses that are in contact with contraction waves at both ends is possible. As the flow rate rises the urine begins to leak through the contraction waves and steady peristaltic flow breaks down. There is an upper limit to the mean flow rate that can be carried by steady peristalsis, which depends on the mechanical properties of the ureter. At high flow rates the peristaltic contractions do not pump but hinder the flow of urine through the ureter.     

The avian oesophageal motor function and its nervous control: some physiological, pharmacological and comparative aspects.

1. This paper deals with the avian oesophageal motor function and it attempts to draw some comparative aspects between neural regulation of the avian and mammalian oesophagus. 2. Different from the mammalian oesophagus, the avian oesophagus, presents at rest electrical activity associated to spontaneous contractions. 3. Swallowing elicits peristaltic contraction, characterized by an inhibitory and an excitatory component. 4. Non-adrenergic, non-cholinergic neurons are responsible for the inhibitory component. 5. Contrarily to what observed in mammals, where the peripheral mechanism are important for the peristaltic sequence, the primary peristaltism of birds seems to be entirely mediated by extrinsic nervous system.     

Muscular tunic of the dog ureter in the normal state and in ureterohydronephrosis (data of scanning electron microscopy)

By means of scanning electron microscopy and macro- microscopical methods in 16 mongrel dogs the ureters have been studied, normal and at ureterohydronephrosis. The ureteral muscles are spirally shaped. The external muscular layer is oriented along the spiral, its step approximately corresponds to the length of the peristaltic wave. The spiral-shaped course of the muscular fasciculi in the middle layer is of opposite direction, the spiral step corresponds to the ureter diameter. The internal muscular layer is formed as a result of changes in the spiralization angle of the middle muscular layer from outside into inside. Fibrills of the connective tissue framework possess predominantly longitudinal orientation. At ureterohydronephrosis, together with increasing diameter of the ureter, the step of the muscular spiral decreases, the connective tissue fibrillar framework acquires a net-like structure.     

Ureteral motility in sheep

Ureteral motility was studied in twenty-five sodium pentobarbital-anaesthetized sheep. Mean frequency of the peristaltic waves was 15 per min and the range was 11-19. Frequency was the same throughout the length of the ureter. Mean contraction pressure (cm H2O) was 40 in the upper ureter, 35 in the middle ureter and 31 in the lower ureter. Mean concentration time was 1 sec and range was 0.6-1.5. Mean relaxation time was 1.1 sec and range was 0.7-1.5. Diuresis induced by rapid intravenous administration of physiologic sodium chloride solution abolished the peristaltic activity.     

Rhythmic activities of isolated and clustered pacemaker neurons and photoreceptors of Aplysia retina in culture

Each eye of Aplysia contains a circadian clock that produces a robust rhythm of optic nerve impulse activity. To isolate the pacemaker neurons and photoreceptors of the eye and determine their participation in the circadian clock and its generation of rhythmic autoactivity, the retina was dissociated and its cells were placed in primary cell culture. The isolated neurons and photoreceptors survived and vigorously extended neurites tipped with growth cones. Many of the photoreceptors previously described from histological sections of the intact retina were identified in culture, including the large R-type photoreceptor, which gave robust photoresponses, and the smaller tufted, whorled, and flared photoreceptors. The pacemaker neurons responsible for the rhythmic impulse activity generated by the eye were identified by their distinctive monopolar morphology and recordings were made of their activity. Isolated pacemaker neurons produced spontaneous action potentials in darkness, and pacemaker neurons attached to fragments of retina or in an isolated cluster interacted to produce robust spontaneous activity. This study establishes that isolated retinal pacemaker neurons retain their innate autoactivity and ability to produce action potentials in culture and that clusters of coupled pacemaker neurons are capable of generating robust autoactivity comparable to pacemaker neuron rhythmic activity recorded in the intact retina, which was previously shown to correspond to 1:1 with the optic nerve compound action potential activity. © 1996 John Wiley & Sons, Inc.     

Interstitial cells of Cajal in the urethra

The smooth muscle layer of the urethra generates spontaneous myogenic tone that is thought to make a major contribution to urinary continence. The mechanisms underlying generation of tone remain unclear, however recent studies from our laboratory highlighted a role for a specialised population of pacemaker cells which we originally referred to as interstitial cells (IC) and now term ICC. Urethra ICC possess an electrical pacemaker mechanism characterised by rhythmic activation of Ca(2+)-activated Cl(-) channels leading to spontaneous transient inward currents (STICs) under voltage clamp and spontaneous transient depolarisations (STDs) under current clamp conditions. Both STICS and STDs are now known to be associated with spontaneous Ca(2+) oscillations that result from a complex interplay between release of Ca(2+) from intracellular stores and Ca(2+) influx across the plasma membrane. In this review we will consider some of the precise mechanisms involved in the generation of pacemaker activity and discuss how these are modulated by excitatory and inhibitory neurotransmitters.     

Fstl1 antagonizes BMP signaling and regulates ureter development

Bone morphogenetic protein (BMP) signaling pathway plays important roles in urinary tract development although the detailed regulation of its activity in this process remains unclear. Here we report that follistatin-like 1 (Fstl1), encoding a secreted extracellular glycoprotein, is expressed in developing ureter and antagonizes BMP signaling activity. Mouse embryos carrying disrupted Fstl1 gene displayed prominent hydroureter arising from proximal segment and ureterovesical junction defects. These defects were associated with significant reduction in ureteric epithelial cell proliferation at E15.5 and E16.5 as well as absence of subepithelial ureteral mesenchymal cells in the urinary tract at E16.5 and E18.5. At the molecular level, increased BMP signaling was found in Fstl1 deficient ureters, indicated by elevated pSmad1/5/8 activity. In vitro study also indicated that Fstl1 can directly bind to ALK6 which is specifically expressed in ureteric epithelial cells in developing ureter. Furthermore, Sonic hedgehog (SHH) signaling, which is crucial for differentiation of ureteral subepithelial cell proliferation, was also impaired in Fstl1(-/-) ureter. Altogether, our data suggest that Fstl1 is essential in maintaining normal ureter development by antagonizing BMP signaling.     

Differential gene expression in the developing mouse ureter

In many instances, kidney dysgenesis results as a secondary consequence to defects in the development of the ureter. Through the use of mouse genetics a number of genes associated with such malformations have been identified, however, the cause of many other abnormalities remain unknown. In order to identify novel genes involved in ureter development we compared gene expression in embryonic day (E) 12.5, E15.5 and postnatal day (P) 75 ureters using the Compugen mouse long oligo microarrays. A total of 248 genes were dynamically upregulated and 208 downregulated between E12.5 and P75. At E12.5, when the mouse ureter is comprised of a simple cuboidal epithelium surrounded by ureteric mesenchyme, genes previously reported to be expressed in the ureteric mesenchyme, foxC1 and foxC2 were upregulated. By E15.5 the epithelial layer develops into urothelium, impermeable to urine, and smooth muscle develops for the peristaltic movement of urine towards the bladder. The development of these two cell types coincided with the upregulation of UPIIIa, RAB27b and PPARgamma reported to be expressed in the urothelium, and several muscle genes, Acta1, Tnnt2, Myocd, and Tpm2. In situ hybridization identified several novel genes with spatial expression within the smooth muscle, Acta1; ureteric mesenchyme and smooth muscle, Thbs2 and Col5a2; and urothelium, Kcnj8 and Adh1. This study marks the first known report defining global gene expression of the developing mouse ureter and will provide insight into the molecular mechanisms underlying kidney and lower urinary tract malformations.     

Bimodal septal and cortical triggering and complex propagation patterns of spontaneous waves of activity in the developing mouse cerebral cortex

Spontaneous waves of activity that propagate across large structures during specific developmental stages play central roles in CNS development. To understand the genesis and functions of these waves, it is critical to understand the spatial and temporal patterns of their propagation. We recently reported that spontaneous waves in the neonatal cerebral cortex originate from a ventrolateral pacemaker region. We have now analyzed a large number of spontaneous waves using calcium imaging over the entire area of coronal slices from E18‐P1 mouse brains. In all waves, the first cortical region active is this ventrolateral pacemaker. In half of the waves, however, the cortical pacemaker activity is itself triggered by preceding activity in the septal nuclei. Most waves are restricted to the septum and/or ventral cortex, with only some invading the dorsal cortex or the contralateral hemisphere. Waves fail to propagate at very stereotyped locations at the boundary between ventral and dorsal cortex and at the dorsal midline. Waves that cross these boundaries pause at these same locations. Waves at these stages are blocked by both picrotoxin and CNQX, indicating that both GABA_A and AMPA receptors are involved in spontaneous activity. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 70: 679–692, 2010     

Spontaneous Activity in Crustacean Neurons

Single units which discharged with regular spontaneous rhythms without intentional stimulation were observed in the ventral nerve cord by intracellular recording close to the sixth abdominal ganglion. These units were divided into two groups: group A units in which interspike intervals varied less than 10 msec.; group B units in which interspike intervals varied within a range of 10 to 30 msec. Group A units maintained "constant" interspike intervals and could not be discharged by sensory inputs, while the majority of group B units could be discharged by appropriate sensory nerve stimulation. Both group A and B units discharged to direct stimulation when the stimulating and recording electrodes were placed in the same ganglionic intersegment, and directly evoked single spikes reset the spontaneous rhythm. In group B units, presynaptic volleys reset the spontaneous rhythm of some units; but in others, synaptically evoked spikes were interpolated within the spontaneous rhythm without resetting. The phenomenon of enhancement could also be demonstrated in spontaneously active units as a result of repetitive stimulation. It is concluded that endogenous pacemaker activity is responsible for much of the regular spontaneous firing observed in crayfish central neurons, and that interaction of evoked responses with such pacemaker sites can produce a variety of effects dependent upon the anatomical relationships between pacemaker and synaptic regions.