William J. Hatton

Analysis of cell death in the trochlear nucleus of the chick embryo: calibration of the optical disector counting method reveals systematic bias.

Detection of changes in numbers of neurons is essential for an understanding of neuronal development, function, and death. Optical disector counting is claimed to be the most efficient technique to estimate accurate numbers of neurons in microscopic sections. We calibrated the optical disector by comparison with three‐dimensional reconstructions from serial sections and determined how accurate this technique is relative to conventional profile counting methods. The calibration was performed on the trochlear nucleus in developing chicks. Optical disector estimates, when obtained as generally recommended, were about 25% lower than the actual number of neurons. This underestimate was caused by a nonuniform (bimodal) distribution of neuronal nuclei in paraffin and plastic (glycolmethacrylate) sections, but not in cryosections. The density of neurons in the core of the paraffin and plastic sections was substantially lower than in the upper and lower margins of these sections. Accurate estimates of neuronal numbers were obtained with a modified optical disector method that sampled the entire extent of tissue sections. Previous estimates of numbers of trochlear neurons in the developing chick have been controversial. The modified (calibrated) optical disector method revealed that the number of trochlear neurons decreased from about 1,600 at day 8.5 of incubation (embryonic day, [E]8.5) to about 900 at the time of hatching. Numbers of pyknotic nuclei peaked at E6 and at E9, revealing an additional early, but postproliferative, period of cell death. Taken together, these data emphasize the need for calibration of stereological counting techniques and the need to examine sampling strategies for potential bias. J. Comp. Neurol. 409:169–186, 1999.

Differential tissue shrinkage and compression in the z-axis: implications for optical disector counting in vibratome-, plastic- and cryosections

The optical disector is among the most efficient cell counting methods, but its accuracy depends on an undistorted particle distribution in the z-axis of tissue sections. Because the optical disector samples particle densities exclusively in the center of sections, it is essential for unbiased estimates of particle numbers that differential shrinkage or compression (and resulting differences in particle densities along the z-axis) are known and corrected. Here we examined, quantified, and compared differential shrinkage and compression of vibratome-, celloidin- and cryosections. Vibratome sections showed a significant z-axis distortion, while celloidin- and cryosections were minimally distorted. Results were directly compared with previous data obtained from paraffin and methacrylate sections. We conclude that z-axis distortion varies significantly between embedding and sectioning methods, and that vibratome-, methacrylate- and paraffin sections can result in grossly biased estimates. We describe a simple method for assessing differential z-axis shrinkage or compression, as well as simple strategies to minimize the bias of the optical disector. Minimal bias can be achieved by either adjusting the placement and extent of counting boxes and guard spaces for sampling, or by applying a correction factor in cases when guard spaces are deemed essential for particle recognition.

Altered properties of volume‐sensitive osmolyte and anion channels (VSOACs) and membrane protein expression in cardiac and smooth muscle myocytes from Clcn3‐/‐ mice

ClC‐3, a member of the large superfamily of ClC voltage‐dependent Cl– channels, has been proposed as a molecular candidate responsible for volume‐sensitive osmolyte and anion channels (VSOACs) in some cells, including heart and vascular smooth muscle. However, the reported presence of native VSOACs in at least two cell types from transgenic ClC‐3 disrupted (Clcn3−/−) mice casts considerable doubt on this proposed role for ClC‐3. We compared several properties of native VSOACs and examined mRNA transcripts and membrane protein expression profiles in cardiac and pulmonary arterial smooth muscle cells from Clcn3+/+ and Clcn3−/− mice to: (1) test the hypothesis that native VSOACs are unaltered in cells from Clcn3−/− mice, and (2) test the possibility that targeted inactivation of the Clcn3 gene using a conventional murine global knock‐out approach may result in compensatory changes in expression of other membrane proteins. Our experiments demonstrate that VSOAC currents in myocytes from Clcn3+/+ and Clcn3−/− mice are remarkably similar in terms of activation and inactivation kinetics, steady‐state current densities, rectification, anion selectivity (I− > Cl−≫ Asp−) and sensitivity to block by glibenclamide, niflumic acid, DIDS and extracellular ATP. However, additional experiments revealed several significant differences in other fundamental properties of native VSOACs recorded from atrial and smooth muscle cells from Clcn3−/− mice, including: differences in regulation by endogenous protein kinase C, differential sensitivity to block by anti‐ClC‐3 antibodies, and differential sensitivities to [ATP]i and free [Mg2+]i. These results suggest that in response to Clcn3 gene deletion, there may be compensatory changes in expression of other proteins that alter VSOAC channel subunit composition or associated regulatory subunits that give rise to VSOACs with different properties. Consistent with this hypothesis, in atria from Clcn3−/− mice compared to Clcn3+/+ mice, quantitative analysis of ClC mRNA expression levels revealed significant increases in transcripts for ClC‐1, ClC‐2, and ClC‐3, and protein expression profiles obtained using two‐dimensional polyacrylamide gel electrophoresis revealed complex changes in at least 35 different unidentified membrane proteins in cells from Clcn3−/− mice. These findings emphasize that caution needs to be exercised in simple attempts to interpret the phenotypic consequences of conventional global Clcn3 gene inactivation.

Functional inhibition of native volume-sensitive outwardly rectifying anion channels in muscle cells and Xenopus oocytes by anti-ClC-3 antibody

1 Intracellular dialysis of NIH/3T3 cells with a commercially available anti‐ClC‐3 polyclonal antibody (Ab) for ≈30 min completely inhibited expressed guinea‐pig ClC‐3 currents (IgpClC‐3), while intracellular dialysis with antigen‐preabsorbed anti‐ClC‐3 Ab failed to affect IgpClC‐3. 2 Anti‐ClC‐3 Ab was used as a selective probe to examine the relationship between endogenous ClC‐3 expression and native volume‐sensitive outwardly rectifying anion channels (VSOACs) in guinea‐pig cardiac cells, canine pulmonary arterial smooth muscle cells (PASMCs) and Xenopus laevis oocytes. Intracellular dialysis or injection of anti‐ClC‐3 Ab abolished native VSOAC function in cardiac cells and PASMCs and significantly reduced VSOACs in oocytes. In contrast, native VSOAC function was unaltered by antigen‐preabsorbed anti‐ClC‐3 Ab. 3 It is suggested that endogenous ClC‐3 represents a major molecular entity responsible for native VSOACs in cardiac and smooth muscle cells and Xenopus oocytes. Anti‐ClC‐3 Ab should be a useful experimental tool to directly test the relationship between endogenous ClC‐3 expression and native VSOAC function, and help resolve existing controversies related to the regulation and physiological role of native VSOACs in a wide variety of different cells.

Functional and molecular expression of a voltage-dependent K+ channel (Kv1.1) in interstitial cells of Cajal

Located within the gastrointestinal (GI) musculature are networks of cells known as interstitial cells of Cajal (ICC). ICC are associated with several functions including pacemaker activity that generates electrical slow waves and neurotransmission regulating GI motility. In this study we identified a voltage‐dependent K+ channel (Kv1.1) expressed in ICC and neurons but not in smooth muscle cells. Transcriptional analyses demonstrated that Kv1.1 was expressed in whole tissue but not in isolated smooth muscle cells. Immunohistochemical co‐localization of Kv1.1 with c‐kit (a specific marker for ICC) and vimentin (a specific marker of neurons and ICC) indicated that Kv1.1‐like immunoreactivity (Kv1.1‐LI) was present in ICC and neurons of GI tissues of the dog, guinea‐pig and mouse. Kv1.1‐LI was not observed in smooth muscle cells of the circular and longitudinal muscle layers. Kv1.1 was cloned from a canine colonic cDNA library and expressed in Xenopus oocytes. Pharmacological investigation of the electrophysiological properties of Kv1.1 demonstrated that the mamba snake toxin dendrotoxin‐K (DTX‐K) blocked the Kv1.1 outward current when expressed as a homotetrameric complex (EC50= 0.34 nm). Other Kv channels were insensitive to DTX‐K. When Kv1.1 was expressed as a heterotetrameric complex with Kv1.5, block by DTX‐K dominated, indicating that one or more subunits of Kv1.1 rendered the heterotetrameric channel sensitive to DTX‐K. In patch‐clamp experiments on cultured murine fundus ICC, DTX‐K blocked a component of the delayed rectifier outward current. The remaining, DTX‐insensitive current (i.e. current in the presence of 10−8m DTX‐K) was outwardly rectifying, rapidly activating, non‐inactivating during 500 ms step depolarizations, and could be blocked by both tetraethylammonium (TEA) and 4‐aminopyridine (4‐AP). In conclusion, Kv1.1 is expressed by ICC of several species. DTX‐K is a specific blocker of Kv1.1 and heterotetrameric channels containing Kv1.1. This information is useful as a means of identifying ICC and in studies of the role of delayed rectifier K+ currents in ICC functions.

Role for the α7β1 integrin in vascular development and integrity

The α7β1 integrin is a laminin receptor that has been implicated in muscle disease and the development of neuromuscular and myotendinous junctions. Studies have shown the α7β1 integrin is also expressed in nonskeletal muscle tissues. To identify the expression pattern of the α7 integrin in these tissues during embryonic development, α7 integrin chain knockout mice were generated by a LacZ knockin strategy. In these mice, expression from the α7 promoter is reported by β‐galactosidase. From embryonic day (ED) 11.5 to ED14.5, β‐galactosidase was detected in the developing central and peripheral nervous systems and vasculature. The loss of the α7 integrin gene resulted in partial embryonic lethality. Several α7 null embryos were identified with cerebrovascular hemorrhages and showed reduced vascular smooth muscle cells and cerebral vascularization. The α7 null mice that survived to birth exhibited vascular smooth muscle defects, including hyperplasia and hypertrophy. In addition, altered expression of α5 and α6B integrin chains was detected in the cerebral arteries of α7 null mice, which may contribute to the vascular phenotype. Our results demonstrate for the first time that the α7β1 integrin is important for the recruitment or survival of cerebral vascular smooth muscle cells and that this integrin plays an important role in vascular development and integrity. Developmental Dynamics 234:11–21, 2005.

ClC‐3 chloride channel is upregulated by hypertrophy and inflammation in rat and canine pulmonary artery

1 Cl− channels have been implicated in essential cellular functions including volume regulation, progression of cell cycle, cell proliferation and contraction, but the physiological functions of the ClC‐3 channel are controversial. We tested the hypothesis that the ClC‐3 gene (ClCn‐3) is upregulated in hypertensive pulmonary arteries of monocrotaline‐treated rats, and upregulated ClC‐3 channel aids viability of pulmonary artery smooth muscle cells (PASMCs). 2 Experimental pulmonary hypertension was induced in rats by a single subcutaneous administration of monocrotaline (60 mg kg−1). Injected animals developed characteristic features of pulmonary hypertension including medial hypertrophy of pulmonary arteries and right ventricular hypertrophy. 3 Reverse transcriptase–polymerase chain reaction (RT–PCR), immunohistochemistry and Western immunoblot analysis indicated that histopathological alterations were associated with upregulation of the ClC‐3 mRNA and protein expression in both smooth muscle cells of hypertensive pulmonary arteries and in cardiac myocytes. 4 RT–PCR analysis of mRNA, extracted from canine cultured PASMCs, indicated that incubation with the inflammatory mediators endothelin‐1 (ET‐1), platelet‐derived growth factor (PDGF), interleukin‐1beta (IL‐1β) and tumor necrosis factor alpha (TNFα), but not transforming growth factor beta (TGFβ), upregulated ClC‐3 mRNA. 5 Adenovirus‐mediated delivery and overexpression of ClC‐3 in canine PASMCs improved cell viability against increasing concentrations of hydrogen peroxide (H2O2, range 50–250 μM). 6 In conclusion, upregulation of ClC‐3 in rat hypertensive lung and heart is a novel observation. Our functional data suggest that upregulation of ClC‐3 is an adaptive response of inflamed pulmonary artery, which enhances the viability of PASMCs against reactive oxygen species.

Platelet-derived growth factor receptor-α cells in mouse urinary bladder: a new class of interstitial cells

Specific classes of interstitial cells exist in visceral organs and have been implicated in several physiological functions including pacemaking and mediators in neurotransmission. In the bladder, Kit+ interstitial cells have been reported to exist and have been suggested to be neuromodulators. More recently a second interstitial cell, which is identified using antibodies against platelet‐derived growth factor receptor‐α (PDGFR‐α) has been described in the gastrointestinal (GI) tract and has been implicated in enteric motor neurotransmission. In this study, we examined the distribution of PDGFR‐α+ cells in the murine urinary bladder and the relation that these cells may have with nerve fibres and smooth muscle cells. Platelet‐derived growth factor receptor‐α+ cells had a spindle shape or stellate morphology and often possessed multiple processes that contacted one another forming a loose network. These cells were distributed throughout the bladder wall, being present in the lamina propria as well as throughout the muscularis of the detrusor. These cells surrounded and were located between smooth muscle bundles and often came into close morphological association with intramural nerve fibres. These data describe a new class of interstitial cells that express a specific receptor within the bladder wall and provide morphological evidence for a possible neuromodulatory role in bladder function.

Novel regulation of the A-type K+ current in murine proximal colon by calcium-calmodulin-dependent protein kinase II

1 The kinetics of inactivation of delayed rectifier K+ current in murine colonic myocytes differed in amphotericin‐permeabilized patch and conventional patch clamp. The difference was accounted for by Ca2+ buffering. 2 Calcium‐calmodulin‐dependent protein kinase II (CaMKII) inhibitors increased the rate of inactivation and slowed recovery from inactivation of the outward current. This was seen in single steps and in the envelope of the current tails. The effect was largely on the TEA‐insensitive component of current. 3 Dialysis of myocytes with autothiophosphorylated CaMKII slowed inactivation. This effect was reversed by addition of CaMKII inhibitor. 4 Antibodies revealed CaMKII‐like immunoreactivity in murine colonic myocytes and other cells. Immunoblots identified a small protein with CaMKII‐like immunoreactivity in homogenates of colonic muscle. 5 We conclude that CaMKII regulates delayed rectifier K+ currents in murine colonic myocytes. The changes in the delayed rectifier current may participate in the Ca2+‐dependent regulation of gastrointestinal motility.

Expression, localization, and functional properties of Bestrophin 3 channel isolated from mouse heart

Bestrophins are a novel family of proteins that encode calcium-activated chloride channels. In this study we establish that Bestrophin transcripts are expressed in the mouse and human heart. Native mBest3 protein expression and localization in heart was demonstrated by using a specific polyclonal mBest3 antibody. Immunostaining of isolated cardiac myocytes indicates that mBest3 is present at the membrane. Using the patch-clamp technique, we characterized the biophysical and pharmacological properties of mBest3 cloned from heart. Whole cell chloride currents were evoked in both HEK293 and COS-7 cells expressing mBest3 by elevation of intracellular calcium. mBest3 currents displayed a K(D) for Ca(2+) of approximately 175 nM. The calcium-activated chloride current was found to be time and voltage independent and displayed slight outward rectification. The anion permeability sequence of the channel was SCN(-)>I(-)>Cl(-), and the current was inhibited by niflumic acid and DIDS in the micromolar range. In addition, we generated a site-specific mutation (F80L) in the putative pore region of mBest3 that significantly altered the ion conduction and pharmacology of this channel. Our functional and mutational studies examining the biophysical properties of mBest3 indicate that it functions as a pore-forming chloride channel that is activated by physiological levels of calcium. This study reports novel findings regarding the molecular expression, tissue localization, and functional properties of mBest3 cloned from heart.