Cardiac receptor physiology and its application to clinical imaging: present and future.

Both gamma imaging and positron emission tomography (PET) imaging of cell surface receptors have become possible through the development of agonists and antagonists with high specific radioactivity and high specificity for the receptors. An understanding of the physiology of the cardiac receptor system is essential to comprehending receptor imaging. The complexity of the physiologic information developed over the past decade has been compounded by the concomitant discovery of additional receptor subtypes. The following is a review of a select group of cardiac receptors and their regulation-namely, adrenergic, muscarinic-cholinergic, adenosine, and angiotensin I and II receptors. The role of imaging regional receptor localization and function in providing new insights into cardiac pathology and therapeutic avenues is explored.

Compact myelin dictates the differential targeting of two sodium channel isoforms in the same axon.

Voltage-dependent sodium channels are uniformly distributed along unmyelinated axons, but are highly concentrated at nodes of Ranvier in myelinated axons. Here, we show that this pattern is associated with differential localization of distinct sodium channel alpha subunits to the unmyelinated and myelinated zones of the same retinal ganglion cell axons. In adult axons, Na(v)1.2 is localized to the unmyelinated zone, whereas Na(v)1.6 is specifically targeted to nodes. During development, Na(v)1.2 is expressed first and becomes clustered at immature nodes of Ranvier, but as myelination proceeds, Na(v)1.6 replaces Na(v)1.2 at nodes. In Shiverer mice, which lack compact myelin, Na(v)1.2 is found throughout adult axons, whereas little Na(v)1.6 is detected. Together, these data show that sodium channel isoforms are differentially targeted to distinct domains of the same axon in a process associated with formation of compact myelin.

Right ventricular involvement in hypertrophic cardiomyopathy: a case report and literature review.

Although hypertrophic cardiomyopathy (HCM) is classically considered a disease of the left ventricle, right ventricular (RV) abnormalities have also been reported. However, involvement of the right ventricle in HCM has not been extensively characterized. The literature regarding prevalence, genetics, patterns of involvement, histologic findings, symptoms, diagnosis, and treatment of RV abnormalities in HCM is reviewed. To highlight the salient points, a case is presented of apical HCM with significant RV involvement, with an RV outflow tract gradient and near obliteration of the RV cavity, in the absence of a left intraventricular gradient. Right ventricular involvement in HCM appears to be as heterogeneous as that of the left ventricle. The spectrum extends from mild concentric hypertrophy to more unusual severe, obstructive disease. While in some cases the extent of RV involvement correlates with left ventricular (LV) involvement, predominant RV disease can be seen as well. While the genetics of RV involvement have not been well characterized, histologic findings appear to be similar to those in the left ventricle, suggesting similar pathogenesis. Significant RV involvement may result in RV outflow obstruction and/or reduced RV diastolic filling, with potentially increased incidence of severe dyspnea, supraventricular arrhythmias, and pulmonary thromboembolism. The optimal treatment for patients with significant RV disease is unknown. Medical and surgical therapies have been attempted with variable success; experience with newer techniques such as percutaneous catheter ablation has not been reported. Further characterization of RV involvement in HCM is necessary to elucidate more clearly the clinical features and optimal treatments of this manifestation of HCM.

GOLAC: an endogenous anion channel of the Golgi complex.

The Golgi complex is present in every eukaryotic cell and functions in posttranslational modifications and sorting of proteins and lipids to post-Golgi destinations. Both functions require an acidic lumenal pH and transport of substrates into and by-products out of the Golgi lumen. Endogenous ion channels are expected to be important for these features, but none has been described. Ion channels from an enriched Golgi fraction cleared of transiting proteins were incorporated into planar lipid bilayers. Eighty percent of the single-channel recordings revealed the same anion channel. This channel has novel properties and has been named GOLAC (Golgi anion channel). The channel has six subconductance states with a maximum conductance of 130 pS, is open over 95% of the time, and is not voltage-gated. Significant for Golgi function, the channel conductance is increased by reduction of pH on the lumenal surface. This channel may serve two nonexclusive functions: providing counterions for the acidification of the Golgi lumen by the H(+)-ATPase and removal of inorganic phosphate generated by glycosylation and sulfation of proteins and lipids in the Golgi.

Sodium channel Na(v)1.6 is localized at nodes of ranvier, dendrites, and synapses.

Voltage-gated sodium channels perform critical roles for electrical signaling in the nervous system by generating action potentials in axons and in dendrites. At least 10 genes encode sodium channels in mammals, but specific physiological roles that distinguish each of these isoforms are not known. One possibility is that each isoform is expressed in a restricted set of cell types or is targeted to a specific domain of a neuron or muscle cell. Using affinity-purified isoform-specific antibodies, we find that Na(v)1.6 is highly concentrated at nodes of Ranvier of both sensory and motor axons in the peripheral nervous system and at nodes in the central nervous system. The specificity of this antibody was also demonstrated with the Na(v)1.6-deficient mouse mutant strain med, whose nodes were negative for Na(v)1.6 immunostaining. Both the intensity of labeling and the failure of other isoform-specific antibodies to label nodes suggest that Na(v)1.6 is the predominant channel type in this structure. In the central nervous system, Na(v)1.6 is localized in unmyelinated axons in the retina and cerebellum and is strongly expressed in dendrites of cortical pyramidal cells and cerebellar Purkinje cells. Ultrastructural studies indicate that labeling in dendrites is both intracellular and on dendritic shaft membranes. Remarkably, Na(v)1.6 labeling was observed at both presynaptic and postsynaptic membranes in the cortex and cerebellum. Thus, a single sodium channel isoform is targeted to different neuronal domains and can influence both axonal conduction and synaptic responses.

Clustering of sodium channels at the neuromuscular junction.

Voltage-gated sodium channels (NaChs) are highly concentrated in the postsynaptic region of the neuromuscular junction, especially in the depths of postsynaptic folds and in the perijunctional region. The formation of the high NaCh density occurs during synapse maturation, approximately 2 weeks after initial synaptic contact in the rodent. The concentration of NaChs and their localization in the troughs of the folds increase the safety factor for neuromuscular transmission by reducing the threshold for initiation of the action potential. There is evidence that agrin plays a role in the formation of NaCh aggregation. Molecules such as ankyrin and syntrophin that bind NaChs may be important for maintenance of the high channel density at the endplate.

Immunolocalization of sodium channel isoform NaCh6 in the nervous system.

Sodium channel 6 (NaCh6) is the alpha-subunit of a voltage-gated sodium channel expressed in the rat nervous system. The mRNA for this isoform has been shown to be expressed in both neuronal and glial cells by in situ hybridization. To examine localization of NaCh6 protein, polyclonal antibodies specific for NaCh6 were generated against peptides from two cytoplasmic domains and a fusion protein from an extracellular domain. Affinity-purified antibodies were used to localize NaCh6 in the brain, spinal cord, peripheral nervous system, and neuromuscular junction. There was widespread labeling of neurons in the brain and spinal cord. NaCh6 was present in both sensory and motor pathways. Radial glial cells in the cerebellum were intensely labeled for both GFAP and NaCh6. At the subcellular level, NaCh6 is found in axons, dendrites, and the cell body. Motor neurons and primary sensory neurons in dorsal root ganglia had strong cytoplasmic and axonal staining. Nodes of Ranvier in peripheral nerve and in the spinal cord were also intensely labeled. Motor neuron axons near the neuromuscular junction were labeled up to, but not including, terminal boutons. Dendrites of pyramidal cells in the cortex, hippocampus, and cerebellum were labeled. NaCh6 is the first NaCh subtype to be localized either at the node of Ranvier or to a dendrite. We conclude that NaCh6 is widely distributed in the central and peripheral nervous systems and is likely to be important for the electrical properties of the axon and dendrite.

Developmental and regional expression of sodium channel isoform NaCh6 in the rat central nervous system.

The sodium channel isoform NaCh6 is abundant in the adult rat brain and is expressed in both neurons and glia (Schaller et al. [1995] J. Neurosci. 15:3231-3242; Krzemien et al. [2000] J. Comp. Neurol. 20:70-83). With reverse transcriptase-polymerase chain reaction (RT-PCR), in situ hybridization, and immunolabeling, NaCh6 expression was investigated in the developing rat brain and spinal cord [embryonic day 15 (E15) through postnatal day 28 (P28)]. The relative abundance of the four major central nervous system NaCh subtypes was quantitated with RT-PCR. In all regions that were investigated (olfactory bulb, cortex, hippocampus, cerebellum, and spinal cord), each subtype had a unique pattern of expression. NaCh6 mRNA and protein were not detected in either brain or spinal cord at E15 and E18 by in situ hybridization and immunohistochemistry. Neurons in the hippocampus, cortex, and olfactory bulb began to express NaCh6 mRNA and protein shortly after birth. The mRNA signal peaked at P7-P14, and protein expression increased as development proceeded. NaCh6 mRNA was detected at P1 in the cerebellum, and a nonuniform distribution of NaCh6 immunoreactivity in both Purkinje cells and granule cells was observed by P7-P14. NaCh6 protein was expressed in granule cells as soon as they left the proliferative phase and began to migrate. Both NaCh6 mRNA and protein were detected in the spinal cord at P1 and were expressed clearly at P7 in motor neurons. The time course of appearance of NaCh6 in postnatal development is consistent with the development of neurologic symptoms in med and jolting mice, which have mutations in the mouse ortholog of NaCh6.

Immunocytochemical localization of NaCh6 in cultured spinal cord astrocytes.

The expression of the alpha-subunit of voltage-gated sodium channel 6 (NaCh6) was examined in cultures of astrocytes from E18 rat spinal cord by using an antibody specific for NaCh6. Stellate cells with processes and flat, pancake-like astrocytes are the two morphological types predominantly present in these cultures. The antibody to NaCh6 labeled clusters at the cell body and along the length of the processes in stellate, process-bearing cells. Weak staining was observed in the flat, pancake-like astrocytes. Together with previous studies (Black et al., Mol Brain Res 23:235-245, 1994, Glia 14:133-144, 1995) that show that stellate cells express NaChs II and III (but not NaCh I) and flat cells express NaCh II, these results support the conclusions that there are different patterns of sodium channel expression between flat and stellate astrocytes and that multiple channel isoforms are expressed within the same cell. This study also suggests that NaCh6 may contribute to the electrical properties found in stellate astrocytes.

Quantitation of presynaptic cardiac sympathetic function with carbon-11-meta-hydroxyephedrine.

UNLABELLED: The purpose of this study was to validate an axially distributed blood-tissue exchange model for the quantitation of cardiac presynaptic sympathetic nervous system function that could be applied to PET images. The model accounts for heterogeneity in myocardial blood flow, differences in transport rates of 11C-meta-hydroxyephedrine (mHED) across the capillary endothelium and/or neuronal membranes, the virtual volumes of distribution in the interstitial space and neuron and retention of mHED in the neuronal vesicles. METHODS: Multiple indicator outflow dilution and residue detection methods were used to measure the kinetics of radiolabeled intravascular space and interstitial space markers and 11C-mHED in isolated perfused rat heart at baseline and during norepinephrine neuronal transporter blockade with desipramine (DMI). The outflow dilution and residue detection data were modeled with a multiple pathway, four-region, axially distributed model of blood-tissue exchange describing flow in the capillary and exchange between regions using permeability-surface area products with units of clearance of milliliters per minute per gram. Meta-hydroxyephedrine may enter the nerve terminal via membrane transport, where it may be sequestered by first-order unidirectional uptake within vesicles. Release of mHED from the vesicles is modeled via exchange with the interstitial space. RESULTS: After intracoronary injection, mHED transport across the capillary endothelium and in the interstitial space closely followed that of sucrose. Subsequently, mHED was retained in the heart, whereas sucrose washed out rapidly. With DMI the outflow dilution curves more closely resembled those of sucrose. Model parameters reflecting capillary-interstitial kinetics and volumes of distribution were unchanged by DMI, whereas parameters reflecting the neuronal transporter process and volumes of distribution in the nerve terminal and vesicular sequestration were markedly decreased by DMI. Application of the model to a pilot set of canine PET images of mHED suggests the feasibility of this approach. CONCLUSION: Meta-hydroxyephedrine kinetics in the heart can be quantitated using an axially distributed, blood-tissue exchange model that accounts for heterogeneity of flow, reflects changes in neuronal function and is applicable to PET images.

Interaction of muscle and brain sodium channels with multiple members of the syntrophin family of dystrophin-associated proteins.

Syntrophins are cytoplasmic peripheral membrane proteins of the dystrophin-associated protein complex (DAPC). Three syntrophin isoforms, alpha1, beta1, and beta2, are encoded by distinct genes. Each contains two pleckstrin homology (PH) domains, a syntrophin-unique (SU) domain, and a PDZ domain. The name PDZ comes from the first three proteins found to contain repeats of this domain (PSD-95, Drosophila discs large protein, and the zona occludens protein 1). PDZ domains in other proteins bind to the C termini of ion channels and neurotransmitter receptors containing the consensus sequence (S/T)XV-COOH and mediate the clustering or synaptic localization of these proteins. Two voltage-gated sodium channels (NaChs), SkM1 and SkM2, of skeletal and cardiac muscle, respectively, have this consensus sequence. Because NaChs are sarcolemmal components like syntrophins, we have investigated possible interactions between these proteins. NaChs copurify with syntrophin and dystrophin from extracts of skeletal and cardiac muscle. Peptides corresponding to the C-terminal 10 amino acids of SkM1 and SkM2 are sufficient to bind detergent-solubilized muscle syntrophins, to inhibit the binding of native NaChs to syntrophin PDZ domain fusion proteins, and to bind specifically to PDZ domains from alpha1-, beta1-, and beta2-syntrophin. These peptides also inhibit binding of the syntrophin PDZ domain to the PDZ domain of neuronal nitric oxide synthase, an interaction that is not mediated by C-terminal sequences. Brain NaChs, which lack the (S/T)XV consensus sequence, also copurify with syntrophin and dystrophin, an interaction that does not appear to be mediated by the PDZ domain of syntrophin. Collectively, our data suggest that syntrophins link NaChs to the actin cytoskeleton and the extracellular matrix via dystrophin and the DAPC.

Fluorine-18-fluoromisonidazole radiation dosimetry in imaging studies.

UNLABELLED: Fluoromisonidazole (FMISO), labeled with the positron emitter 18F, is a useful hypoxia imaging agent for PET studies, with potential applications in patients with tumors, cardiovascular disease and stroke. METHODS: Radiation doses were calculated in patients undergoing imaging studies to help define the radiation risk of FMISO-PET imaging. Time-dependent concentrations of radioactivity were determined in blood samples and PET images of patients following intravenous injection of [18F]FMISO. Radiation absorbed doses were calculated using the procedures of the Medical Internal Radiation Dose (MIRD) committee, taking into account the variation in dose based on the distribution of activities observed in the individual patients. As part of this study we also calculated an S value for brain to eye. Effective dose equivalent was calculated using ICRP 60 weights. RESULTS: Effective dose equivalent was 0.013 mSv/MBq in men and 0.014 mSv/MBq in women. Individual organ doses for women were not different from men. Assuming bladder voiding at 2- or 4-hr intervals, the critical organ that received the highest dose was the urinary bladder wall (0.021 mGy/MBq with 2-hr voiding intervals or 0.029 mGy/MBq with 4-hr voiding intervals). CONCLUSION: The organ doses for [18F]FMISO are comparable to those associated with other commonly performed nuclear medicine tests and indicate that potential radiation risks associated with this study are within generally accepted limits.

High speed liquid chromatography of phenylethanolamines for the kinetic analysis of [11C]-meta-hydroxyephedrine and metabolites in plasma.

A method is developed and described for analysis of [11C]-meta-hydroxyephedrine, [11C]MHED, a tracer of cardiac function, and its metabolites in plasma samples. The method combines on-column solid-phase extraction and separation on a single weak cation-exchange column. Phenylethanolamines were used to develop the separation method that concentrates the analytes on-column from physiological saline and then elutes them by changing to an acidic mobile phase. Hydrophobic interactions determine the selectivity, and elution order is the same as for reversed-phase liquid chromatography on a C1 stationary phase. The mechanism of separation is mixed mode, with ion-exchange coupled with a reversed-phase liquid chromatography mechanism. Each sample analysis requires only 10 min and does not require deproteinization or the use of organic solvents. In human samples, a single plasma metabolite of [11C]MHED along with the parent compound were observed using this method. The method was sufficiently rapid so that in 70 min seven samples were assayed, providing a well-defined time course for MHED and its metabolites in blood. The metabolite concentration increased with time to approximately 85% of the plasma activity 50 min after administration. The results with the developed method are comparable to those described for reversed-phase separations, with the advantage that our method does not require deproteinization, reducing sample analysis time by a factor of two.

Aggregation of sodium channels induced by a postnatally upregulated isoform of agrin.

Agrin is involved in signaling the formation of high concentrations of acetylcholine receptors (AChRs) at the neuromuscular junction (NMJ). There are multiple isoforms of agrin attributable to alternative splicing, and these isoforms are differentially expressed during development and between tissues. The ability to cluster AChRs varies among the agrin isoforms. Sodium channels (NaChs) are also concentrated at the NMJ. We have tested various agrin isoforms for their ability to induce formation of clusters of NaChs. We grew cocultures of dissociated adult rat muscle fibers with chinese hamster ovary (CHO) cells that had been transfected with different isoforms of agrin. Using immunocytochemical techniques, we determined that after 1 d in culture, CHO cells synthesizing the neuronally expressed isoform with an eight amino acid insert (Agrin8) were able to form NaCh clusters at sites of contact between the CHO cell and muscle cell. Clusters of NaChs could be formed anywhere along a muscle fiber, but more clusters were detected close to the endplate where the endogenous level of NaChs was higher. None of the other neuronal-specific agrin isoforms was able to cluster NaChs. Because Agrin8 is the only agrin isoform that is upregulated at birth when NaChs begin to cluster at the NMJ, we conclude that Agrin8 expression by motor neurons is a signal for NaCh clustering at the NMJ during normal development.

Radiographic manifestations of eosinophilic gastroenteritis.

Eosinophilic gastroenteritis (EG) is a rare inflammatory disease of unknown etiology, characterized by focal or diffuse eosinophilic infiltration of the gastrointestinal tract. Although little over 250 cases of EG have been reported in the literature, EG is probably more common than reports in the literature would indicate. A retrospective review of 25 patients with EG along with a review of the literature was done to identify clinical, laboratory, radiographic, and therapeutic features. An allergic disorder was present in 14 (56%) and a peripheral eosinophilia was present in 24 (96%) of our patients. The most common radiographic manifestations of the stomach and small bowel included stenosis and fold thickening, respectively. Thirteen patients had esophageal involvement, with the esophageal stricture being the most common abnormality found in these patients. Steroids produced a good therapeutic result in most patients; the remaining patients responded to cromolyn and/or surgery.

Comparison of fluorine-18-fluorodeoxyglucose and tritiated fluoromisonidazole uptake during low-flow ischemia.

UNLABELLED: Fluorine-18-fluoromisonidazole (FMISO) is trapped in hypoxic but viable canine myocardium. Because of the potential for its use as a marker of myocardial viability, we compared FMISO activity to [18F]fluorodeoxyglucose (FDG) activity in the same myocardial samples from eight dogs subjected to 3 hr of moderate regional myocardial ischemia. METHODS: Tritiated FMISO was injected 15-30 min after onset of regional ischemia (40%-70% reduction in systolic wall thickening) which was maintained for 3 hr. FDG was injected after 2 hr of ischemia. Myocardial blood flow (MBF) was measured by the radiolabeled microsphere technique at the time of each radiotracer injection. At 3 hr of ischemia, the heart was excised and cut into short-axis slices. One slice encompassing both ischemic and normal tissue was cut into 64 samples. FMISO and FDG activity in each sample were normalized to the mean normal zone activity and further expressed as a function of regional MBF. RESULTS: FMISO uptake was consistently greater than FDG uptake, although this was significantly different only for MBF, between 40%-60% of normal. When analyzed relative to endocardial-epicardial location, endocardial FMISO uptake was significantly greater in all hypoperfused samples. CONCLUSION: These results suggest that FMISO is as sensitive as FDG for detecting myocardial ischemia and could be used for identification of viable myocardium.

A novel, abundant sodium channel expressed in neurons and glia.

A novel, voltage-gated sodium channel cDNA, designated NaCh6, has been isolated from the rat central and peripheral nervous systems. RNase protection assays showed that NaCh6 is highly expressed in the brain, and NaCh6 mRNA is as abundant or more abundant than the mRNAs for previously identified rat brain sodium channels. In situ hybridization demonstrated that a wide variety of neurons express NaCh6, including motor neurons in the brainstem and spinal cord, cerebellar granule cells, and pyramidal and granule cells of the hippocampus. RT-PCR and/or in situ hybridization showed that astrocytes and Schwann cells express NaCh6. Thus, this sodium channel is broadly distributed throughout the nervous system and is shown to be expressed in both neurons and glial cells.

Expression and distribution of sodium channels in short- and long-term denervated rodent skeletal muscles.

1. Loose-patch voltage-clamp recordings were made from rat and mouse skeletal muscle fibres denervated for up to 6 weeks. Innervated muscles possessed a Na+ current density of 107 +/- 3.3 mA cm-2 in endplate membrane, and 6.3 +/- 0.6 mA cm-2 in extrajunctional membrane. This high concentration of Na+ channels at the endplate was gradually reduced following denervation. After 6 weeks of denervation, the endplate Na+ channel concentration was reduced by 40-50%, and the density of Na+ channels in extrajunctional membrane was increased by about 30%. 2. The tetrodotoxin (TTX)-resistant form of the Na+ channel appeared after 3 days of denervation and comprised approximately 43% of the endplate Na+ channels 5-6 days after denervation. Subsequently, TTX-resistant Na+ channels were reduced in density to approximately 25% of the postjunctional Na+ channels and remained at this level up to 6 weeks after denervation. 3. RNase protection analysis showed that mRNA encoding the TTX-resistant Na+ channel was virtually absent in innervated muscle, rose > 50-fold after 3 days of denervation, then decreased by 95% 6 weeks after denervation. The density of TTX-resistant Na+ channels correlated qualitatively with changes in mRNA levels. 4. These results suggest that the density of Na+ channels at neuromuscular junctions is maintained by two mechanisms, one influenced by the nerve terminal and the other independent of innervation.

Muscle sodium channel inactivation defect in paramyotonia congenita with the thr1313met mutation.

Mutations of the skeletal muscle sodium (Na) channel have been reported in families with paramyotonia congenita (PC), an autosomal dominant disorder with cold and/or exercise induced stiffness and myotonia. Functional consequences of specific Na channel mutations responsible for PC have not been described. Patch clamp recording of single Na channels were made in cultured myotubes at 22 and 34 degrees C from a PC patient with the thr1313met mutation. Cell-attached and outside-out recordings of mutant PC channels contained long duration and late openings. The mean open time was increased and the ensemble average showed a prolonged inward Na current. This membrane depolarization could cause repetitive action potentials and the clinical syndrome.