Heat-hypersensitive mutants of ryanodine receptor type 1 revealed by microscopic heating.

Thermoregulation is an important aspect of human homeostasis, and high temperatures pose serious stresses for the body. Malignant hyperthermia (MH) is a life-threatening disorder in which body temperature can rise to a lethal level. Here we employ an optically controlled local heat-pulse method to manipulate the temperature in cells with a precision of less than 1 °C and find that the mutants of ryanodine receptor type 1 (RyR1), a key Ca2+ release channel underlying MH, are heat hypersensitive compared with the wild type (WT). We show that the local heat pulses induce an intracellular Ca2+ burst in human embryonic kidney 293 cells overexpressing WT RyR1 and some RyR1 mutants related to MH. Fluorescence Ca2+ imaging using the endoplasmic reticulum-targeted fluorescent probes demonstrates that the Ca2+ burst originates from heat-induced Ca2+ release (HICR) through RyR1-mutant channels because of the channels' heat hypersensitivity. Furthermore, the variation in the heat hypersensitivity of four RyR1 mutants highlights the complexity of MH. HICR likewise occurs in skeletal muscles of MH model mice. We propose that HICR contributes an additional positive feedback to accelerate thermogenesis in patients with MH.

Regulatory mechanisms of ryanodine receptor/Ca2+ release channel revealed by recent advancements in structural studies.

Ryanodine receptors (RyRs) are huge homotetrameric Ca2+ release channels localized to the sarcoplasmic reticulum. RyRs are responsible for the release of Ca2+ from the SR during excitation-contraction coupling in striated muscle cells. Recent revolutionary advancements in cryo-electron microscopy have provided a number of near-atomic structures of RyRs, which have enabled us to better understand the architecture of RyRs. Thus, we are now in a new era understanding the gating, regulatory and disease-causing mechanisms of RyRs. Here we review recent advances in the elucidation of the structures of RyRs, especially RyR1 in skeletal muscle, and their mechanisms of regulation by small molecules, associated proteins and disease-causing mutations.

Genotype-Phenotype Correlations of Malignant Hyperthermia and Central Core Disease Mutations in the Central Region of the RYR1 Channel.

Type 1 ryanodine receptor (RYR1) is a Ca2+ release channel in the sarcoplasmic reticulum of skeletal muscle and is mutated in some muscle diseases, including malignant hyperthermia (MH) and central core disease (CCD). Over 200 mutations associated with these diseases have been identified, and most mutations accelerate Ca2+ -induced Ca2+ release (CICR), resulting in abnormal Ca2+ homeostasis in skeletal muscle. However, it remains largely unknown how specific mutations cause different phenotypes. In this study, we investigated the CICR activity of 14 mutations at 10 different positions in the central region of RYR1 (10 MH and four MH/CCD mutations) using a heterologous expression system in HEK293 cells. In live-cell Ca2+ imaging, the mutant channels exhibited an enhanced sensitivity to caffeine, a reduced endoplasmic reticulum Ca2+ content, and an increased resting cytoplasmic Ca2+ level. The three parameters for CICR (Ca2+ sensitivity for activation, Ca2+ sensitivity for inactivation, and attainable maximum activity, i.e., gain) were obtained by [3 H]ryanodine binding and fitting analysis. The mutant channels showed increased gain and Ca2+ sensitivity for activation in a site-specific manner. Genotype-phenotype correlations were explained well by the near-atomic structure of RYR1. Our data suggest that divergent CICR activity may cause various disease phenotypes by specific mutations.

Nitric oxide-induced calcium release via ryanodine receptors regulates neuronal function.

Mobilization of intracellular Ca(2+) stores regulates a multitude of cellular functions, but the role of intracellular Ca(2+) release via the ryanodine receptor (RyR) in the brain remains incompletely understood. We found that nitric oxide (NO) directly activates RyRs, which induce Ca(2+) release from intracellular stores of central neurons, and thereby promote prolonged Ca(2+) signalling in the brain. Reversible S-nitrosylation of type 1 RyR (RyR1) triggers this Ca(2+) release. NO-induced Ca(2+) release (NICR) is evoked by type 1 NO synthase-dependent NO production during neural firing, and is essential for cerebellar synaptic plasticity. NO production has also been implicated in pathological conditions including ischaemic brain injury, and our results suggest that NICR is involved in NO-induced neuronal cell death. These findings suggest that NICR via RyR1 plays a regulatory role in the physiological and pathophysiological functions of the brain.

Essential Roles of Natural Products and Gaseous Mediators on Neuronal Cell Death or Survival.

Although precise cellular and molecular mechanisms underlying neurodegeneration still remain enigmatic, key factors associated with degenerative disorders, such as glutamate toxicity and oxidative stress, have been recently identified. Accordingly, there has been growing interest in examining the effects of exogenous and endogenous molecules on neuroprotection and neurodegeneration. In this paper, we review recent studies on neuroprotective and/or neurodegenerative effects of natural products, such as caffeic acid and chlorogenic acid, and gaseous mediators, including hydrogen sulfide and nitric oxide. Furthermore, possible molecular mechanisms of these molecules in relation to glutamate signals are discussed. Insight into the pathophysiological role of these molecules will make progress in our understanding of molecular mechanisms underlying neurodegenerative diseases, and is expected to lead to potential therapeutic approaches.

ß-catenin regulates parathyroid hormone/parathyroid hormone-related protein receptor signals and chondrocyte hypertrophy through binding to the intracellular C-terminal region of the receptor.

OBJECTIVE: To investigate the underlying mechanisms of action and functional relevance of ß-catenin in chondrocytes, by examining the role of ß-catenin as a novel protein that interacts with the intracellular C-terminal portion of the parathyroid hormone (PTH)/PTH-related protein (PTHrP) receptor type 1 (PTHR-1). METHODS: The ß-catenin-PTHR-1 binding region was determined with deletion and mutagenesis analyses of the PTHR1 C-terminus, using a mammalian two-hybrid assay. Physical interactions between these 2 molecules were examined with an in situ proximity ligation assay and immunostaining. To assess the effects of gain- and loss-of-function of ß-catenin, transfection experiments were performed to induce overexpression of the constitutively active form of ß-catenin (ca-ß-catenin) and to block ß-catenin activity with small interfering RNA, in cells cotransfected with either wild-type PTHR1 or mutant forms (lacking binding to ß-catenin). Activation of the G protein α subunits G(αs) and G(αq) in the cells was determined by measurement of the intracellular cAMP accumulation and intracellular Ca(2+) concentration, while activation of canonical Wnt pathways was assessed using a TOPflash reporter assay. RESULTS: In differentiated chondrocytes, ß-catenin physically interacted and colocalized with the cell membrane-specific region of PTHR-1 (584-589). Binding of ß-catenin to PTHR-1 caused suppression of the G(αs)/cAMP pathway and enhancement of the G(αq)/Ca(2+) pathway, without affecting the canonical Wnt pathway. Inhibition of Col10a1 messenger RNA (mRNA) expression by PTH was restored by overexpression of ca-ß-catenin, even after blockade of the canonical Wnt pathway, and Col10a1 mRNA expression was further decreased by knockout of ß-catenin (via the Cre recombinase) in chondrocytes from ß-catenin-floxed mice. Mutagenesis analyses to block the binding of ß-catenin to PTHR1 caused an inhibition of chondrocyte hypertrophy markers. CONCLUSION: ß-catenin binds to the PTHR-1 C-tail and switches the downstream signaling pathway from G(αs)/cAMP to G(αq)/Ca(2+), which is a possible mechanism by which chondrocyte hypertrophy may be regulated through the PTH/PTHrP signal independent of the canonical Wnt pathway.

Trans-scale thermal signaling in biological systems.

Biochemical reactions in cells serve as the endogenous source of heat, maintaining a constant body temperature. This process requires proper control; otherwise, serious consequences can arise due to the unwanted but unavoidable responses of biological systems to heat. This review aims to present a range of responses to heat in biological systems across various spatial scales. We begin by examining the impaired thermogenesis of malignant hyperthermia in model mice and skeletal muscle cells, demonstrating that the progression of this disease is caused by a positive feedback loop between thermally driven Ca2+ signaling and thermogenesis at the subcellular scale. After we explore thermally driven force generation in both muscle and non-muscle cells, we illustrate how in vitro assays using purified proteins can reveal the heat-responsive properties of proteins and protein assemblies. Building on these experimental findings, we propose the concept of 'trans-scale thermal signaling'.

[Therapeutic effects of novel type1 ryanodine receptor inhibitor on skeletal muscle diseases].

Type 1 ryanodine receptor (RyR1) plays a key role in Ca2+ release from the sarcoplasmic reticulum (SR) during excitation-contraction coupling of skeletal muscle. Mutations in RyR1 hyperactivate the channel to cause malignant hyperthermia (MH). MH is a serious complication characterized by skeletal muscle rigidity and elevated body temperature in response to commonly used inhalational anesthetics. Thus far, more than 300 mutations in RyR1 gene have been reported in patients with MH. Some heat stroke triggered by exercise or environmental heat stress is also related to MH mutations in the RyR1 gene. The only drug approved for ameliorating the symptoms of MH is dantrolene, which has been first developed in 1960s as a muscle relaxant. However, dantrolene has several disadvantages for clinical use: poor water solubility which makes rapid preparation difficult in emergency situations and long plasma half-life, which causes long-lasting side effects such as muscle weakness. Here we show that a novel RyR1-selective inhibitor, 6,7-(methylenedioxy)-1-octyl-4-quinolone-3-carboxylic acid (Compound 1, Cpd1), effectively rescues MH and heat stroke in new mouse model relevant to MH. Cpd1 has great advantages of higher water solubility and shorter plasma half-life compared to dantrolene. Our data suggest that Cpd1 has the potential to be a promising new candidate for effective treatment of patients carrying RyR1 mutations.

Mice with R2509C-RYR1 mutation exhibit dysfunctional Ca2+ dynamics in primary skeletal myocytes.

Type 1 ryanodine receptor (RYR1) is a Ca2+ release channel in the sarcoplasmic reticulum (SR) of the skeletal muscle and plays a critical role in excitation-contraction coupling. Mutations in RYR1 cause severe muscle diseases, such as malignant hyperthermia, a disorder of Ca2+-induced Ca2+ release (CICR) through RYR1 from the SR. We recently reported that volatile anesthetics induce malignant hyperthermia (MH)-like episodes through enhanced CICR in heterozygous R2509C-RYR1 mice. However, the characterization of Ca2+ dynamics has yet to be investigated in skeletal muscle cells from homozygous mice because these animals die in utero. In the present study, we generated primary cultured skeletal myocytes from R2509C-RYR1 mice. No differences in cellular morphology were detected between wild type (WT) and mutant myocytes. Spontaneous Ca2+ transients and cellular contractions occurred in WT and heterozygous myocytes, but not in homozygous myocytes. Electron microscopic observation revealed that the sarcomere length was shortened to ∼1.7 µm in homozygous myocytes, as compared to ∼2.2 and ∼2.3 µm in WT and heterozygous myocytes, respectively. Consistently, the resting intracellular Ca2+ concentration was higher in homozygous myocytes than in WT or heterozygous myocytes, which may be coupled with a reduced Ca2+ concentration in the SR. Finally, using infrared laser-based microheating, we found that heterozygous myocytes showed larger heat-induced Ca2+ transients than WT myocytes. Our findings suggest that the R2509C mutation in RYR1 causes dysfunctional Ca2+ dynamics in a mutant-gene dose-dependent manner in the skeletal muscles, in turn provoking MH-like episodes and embryonic lethality in heterozygous and homozygous mice, respectively.

A novel RyR1-selective inhibitor prevents and rescues sudden death in mouse models of malignant hyperthermia and heat stroke.

Mutations in the type 1 ryanodine receptor (RyR1), a Ca2+ release channel in skeletal muscle, hyperactivate the channel to cause malignant hyperthermia (MH) and are implicated in severe heat stroke. Dantrolene, the only approved drug for MH, has the disadvantages of having very poor water solubility and long plasma half-life. We show here that an oxolinic acid-derivative RyR1-selective inhibitor, 6,7-(methylenedioxy)-1-octyl-4-quinolone-3-carboxylic acid (Compound 1, Cpd1), effectively prevents and treats MH and heat stroke in several mouse models relevant to MH. Cpd1 reduces resting intracellular Ca2+, inhibits halothane- and isoflurane-induced Ca2+ release, suppresses caffeine-induced contracture in skeletal muscle, reduces sarcolemmal cation influx, and prevents or reverses the fulminant MH crisis induced by isoflurane anesthesia and rescues animals from heat stroke caused by environmental heat stress. Notably, Cpd1 has great advantages of better water solubility and rapid clearance in vivo over dantrolene. Cpd1 has the potential to be a promising candidate for effective treatment of patients carrying RyR1 mutations.

Inositol 1,4,5-trisphosphate signaling maintains the activity of glutamate uptake in Bergmann glia.

The maintenance of synaptic functions is essential for neuronal information processing in the adult brain. Astrocytes express glutamate transporters that rapidly remove glutamate from the extracellular space and they play a critical role in the precise operation of glutamatergic transmission. However, how the glutamate clearance function of astrocytes is maintained remains elusive. Here, we describe a maintenance mechanism for the glutamate uptake capacity of Bergmann glial cells (BGs) in the cerebellum. When inositol 1,4,5-trisphosphate (IP(3) ) signaling was chronically and selectively inhibited in BGs in vivo, the retention time of glutamate around parallel fiber-Purkinje cell synapses was increased. Under these conditions, a decrease in the level of the glutamate/aspartate transporter (GLAST) in BGs was observed. The same effects were observed after chronic in vivo inhibition of purinergic P2 receptors in the cerebellar cortex. These results suggest that the IP(3) signaling cascade is involved in regulating GLAST levels in BGs to maintain glutamate clearance in the mature cerebellum.

Temporal switching and cell-to-cell variability in Ca2+ release activity in mammalian cells.

Genetically identical cells in a uniform external environment can exhibit different phenotypes, which are often masked by conventional measurements that average over cell populations. Although most studies on this topic have used microorganisms, differentiated mammalian cells have rarely been explored. Here, we report that only approximately 40% of clonal human embryonic kidney 293 cells respond with an intracellular Ca(2+) increase when ryanodine receptor Ca(2+) release channels in the endoplasmic reticulum are maximally activated by caffeine. On the other hand, the expression levels of ryanodine receptor showed a unimodal distribution. We showed that the difference in the caffeine sensitivity depends on a critical balance between Ca(2+) release and Ca(2+) uptake activities, which is amplified by the regenerative nature of the Ca(2+) release mechanism. Furthermore, individual cells switched between the caffeine-sensitive and caffeine-insensitive states with an average transition time of approximately 65 h, suggestive of temporal fluctuation in endogenous protein expression levels associated with caffeine response. These results suggest the significance of regenerative mechanisms that amplify protein expression noise and induce cell-to-cell phenotypic variation in mammalian cells.

[Role of skeletal muscle homeostasis of functional food material].

Functional food material, polyamines are considered to be essential for growth factors in virtually all cells. The polyamines putrescine, spermidine and spermine are low molecular weight organic polycations, well known as mediators involved in cell homeostasis. The proposed functions of polyamines are the regulation of ion channels, nucleic acid packaging, signal transduction, cell proliferation, and differentiation, as well as gene expression. In skeletal muscle, regulation of polyamine levels is associated with muscle hypertrophy and atrophy, yet detailed studies are remained to be undergoing. Here, we studied how polyamines may affect the proliferation and/or differentiation of murine myoblast progenitor C2C12 cell line. Upon polyamine treatment of C2C12 cells during induction of myogenic differentiation, the number of myotubes significantly increased. Morphologically, polyamine-treated myotubes exhibited elongated cell body and contained larger amount of nuclei in the cell. On the other hand, the polyamine did not have influence on myoblasts proliferation. Furthermore, compensatory muscle hypertrophy of C57BL6 mice that underwent sciatic nerve transection of the left hindlimb was enhanced by administration of polyamines. Therefore, our study demonstrates that polyamines may play an important role in regulating myogenic differentiation rather than myoblasts proliferation.

Single-cell temperature mapping with fluorescent thermometer nanosheets.

Recent studies using intracellular thermometers have shown that the temperature inside cultured single cells varies heterogeneously on the order of 1°C. However, the reliability of intracellular thermometry has been challenged both experimentally and theoretically because it is, in principle, exceedingly difficult to exclude the effects of nonthermal factors on the thermometers. To accurately measure cellular temperatures from outside of cells, we developed novel thermometry with fluorescent thermometer nanosheets, allowing for noninvasive global temperature mapping of cultured single cells. Various types of cells, i.e., HeLa/HEK293 cells, brown adipocytes, cardiomyocytes, and neurons, were cultured on nanosheets containing the temperature-sensitive fluorescent dye europium (III) thenoyltrifluoroacetonate trihydrate. First, we found that the difference in temperature on the nanosheet between nonexcitable HeLa/HEK293 cells and the culture medium was less than 0.2°C. The expression of mutated type 1 ryanodine receptors (R164C or Y523S) in HEK293 cells that cause Ca2+ leak from the endoplasmic reticulum did not change the cellular temperature greater than 0.1°C. Yet intracellular thermometry detected an increase in temperature of greater than ∼2°C at the endoplasmic reticulum in HeLa cells upon ionomycin-induced intracellular Ca2+ burst; global cellular temperature remained nearly constant within ±0.2°C. When rat neonatal cardiomyocytes or brown adipocytes were stimulated by a mitochondrial uncoupling reagent, the temperature was nearly unchanged within ±0.1°C. In cardiomyocytes, the temperature was stable within ±0.01°C during contractions when electrically stimulated at 2 Hz. Similarly, when rat hippocampal neurons were electrically stimulated at 0.25 Hz, the temperature was stable within ±0.03°C. The present findings with nonexcitable and excitable cells demonstrate that heat produced upon activation in single cells does not uniformly increase cellular temperature on a global basis, but merely forms a local temperature gradient on the order of ∼1°C just proximal to a heat source, such as the endoplasmic/sarcoplasmic reticulum ATPase.

Insights into channel modulation mechanism of RYR1 mutants using Ca2+ imaging and molecular dynamics.

Type 1 ryanodine receptor (RYR1) is a Ca2+ release channel in the sarcoplasmic reticulum in skeletal muscle and plays an important role in excitation-contraction coupling. Mutations in the RYR1 gene cause severe muscle diseases such as malignant hyperthermia (MH), which is a disorder of CICR via RYR1. Thus far, >300 mutations in RYR1 have been reported in patients with MH. However, owing to a lack of comprehensive analysis of the structure-function relationship of mutant RYR1, the mechanism remains largely unknown. Here, we combined functional studies and molecular dynamics (MD) simulations of RYR1 bearing disease-associated mutations at the N-terminal region. When expressed in HEK293 cells, the mutant RYR1 caused abnormalities in Ca2+ homeostasis. MD simulations of WT and mutant RYR1s were performed using crystal structure of the N-terminal domain (NTD) monomer, consisting of A, B, and C domains. We found that the mutations located around the interdomain region differentially affected hydrogen bonds/salt bridges. Particularly, mutations at R402, which increase the open probability of the channel, cause clockwise rotation of BC domains with respect to the A domain by alteration of the interdomain interactions. Similar results were also obtained with artificial mutations that mimic alteration of the interactions. Our results reveal the importance of interdomain interactions within the NTD in the regulation of the RYR1 channel and provide insights into the mechanism of MH caused by the mutations at the NTD.

Primary cultured neuronal networks and type 2 diabetes model mouse fatty liver tissues in aqueous liquid observed by atmospheric SEM (ASEM): Staining preferences of metal solutions.

Neural networking, including axon targeting and synapse formation, is the basis of various brain functions, including memory and learning. Diabetes-mellitus affects peripheral nerves and is known to cause fatty liver disease. Electron microscopy (EM) provides the resolution required to observe changes in fine subcellular structures caused by such physiological and pathological processes, but samples are observed in vacuum. Environmental capsule EM can directly observe cells in a more natural aqueous environment, but the size-limited capsules restrict cell culturability. The recently developed atmospheric scanning electron microscope (ASEM) has an open, 35 mm sample dish, allowing the culture of primary cells, including neurons, on the electron-transparent film window fabricated in its base. The system's inverted scanning electron microscope observes aldehyde-fixed cells or tissues from below through the silicon nitride film; the optical microscope located above allows direct correlation of fluorescence labeling. To observe fixed biological samples, damage due to low dose electron radiation is minimized in three ways. First, knock on damage that pushes out atoms is decreased by the low accelerating voltage of 10-30 kV. Second, increased radical generation due to the decreased acceleration voltage is countered by the addition of a radical scavenger, glucose or ascorbic acid, to the sample solution. Third, the large volume (max. 2 ml) of aqueous buffer surrounding the sample has a high specific heat capacity, minimizing the temperature increase caused by irradiation. Using ASEM, we have developed protocols for heavy metal staining in solution to selectively visualize intracellular structures. Various EM staining methods served as a starting point. Uranyl acetate preferably stains proteins and nucleic acid, and prior tannic acid treatment enhances membranes. Osmium tetroxide is suggested to enhance lipids, especially oil droplets. Imaging primary-culture neurons stained with platinum blue or uranyl acetate revealed growth cones, synapses, and 50-500 nm spines, together with neurite backbones and their associated structures. Correlative microscopy with immuno-fluorescence labeling suggested that these were mainly microtubule associated objects; some showed signs of a fission process and were, thus, possibly mitochondria. Liver tissue excised from the ob/ob type 2 diabetes model mouse, was stained with osmium tetroxide and observed using ASEM. Swollen bright balls occupied a large area of the cytoplasm and could be distinguished from vacuoles, suggesting that they are oil droplets. In some of the images, oil-like droplets were pressing surrounding structures, including sinusoids, significant for blood circulation in the liver. Based on these studies, ASEM combined with metal staining methods promises to allow the study of various mesoscopic-scale phenomena of cells and tissues immersed in natural aqueous environment in the near future. The quick nature of ASEM could facilitate not only the precise imaging for neuroscience but also the diagnosis of fatty liver disease and related diseases.

Correlative light-electron microscopy in liquid using an inverted SEM (ASEM).

In atmospheric scanning electron microscope (ASEM), the inverted scanning electron microscope (SEM) observes the wet sample from below, while an optical microscope observes it from above simultaneously. The ASEM sample holder has a disposable dish shape with a silicon nitride film window at the bottom. It can be coated variously for the primary-culture of substrate-sensitive cells; primary cells were cultured in a few milliliters of culture medium in a stable incubator environment. For the inverted SEM observation, cells and the excised tissue blocks were aldehyde-fixed, immersed in radical scavenger solution, and observed at minimum electron dose. Neural networking, axonal segmentation, proplatelet-formation and phagocytosis, and Fas expression in embryonic stem cells were captured by optical or fluorescence microscopy, and imaged at high resolution by gold-labeled immuno-ASEM with/without metal staining. By exploiting optical microscopy, the region of interest of organ can be found from the wide area, and the cells and organelle were successfully examined at high resolution by the following scanning electron microscopy. We successfully visualized islet of Langerhans, blood microvessels, neuronal endplate, and bacterial flora on stomach epidermal surfaces. Bacterial biofilms and the typical structural features including "leg complex" of mycoplasma were visualized by exploiting CLEM of ASEM. Based on these studies, ASEM correlative microscopy promises to allow the research of various mesoscopic-scale biological phenomena in the near future.

Secretory glands and microvascular systems imaged in aqueous solution by atmospheric scanning electron microscopy (ASEM).

Exocrine glands, e.g., salivary and pancreatic glands, play an important role in digestive enzyme secretion, while endocrine glands, e.g., pancreatic islets, secrete hormones that regulate blood glucose levels. The dysfunction of these secretory organs immediately leads to various diseases, such as diabetes or Sjögren's syndrome, by poorly understood mechanisms. Gland-related diseases have been studied by optical microscopy (OM), and at higher resolution by transmission electron microscopy (TEM) of Epon embedded samples, which necessitates hydrophobic sample pretreatment. Here, we report the direct observation of tissue in aqueous solution by atmospheric scanning electron microscopy (ASEM). Salivary glands, lacrimal glands, and pancreas were fixed, sectioned into slabs, stained with phosphotungstic acid (PTA), and inspected in radical scavenger d-glucose solution from below by an inverted scanning electron microscopy (SEM), guided by optical microscopy from above to target the tissue substructures. A 2- to 3-µm specimen thickness was visualized by the SEM. In secretory cells, cytoplasmic vesicles and other organelles were clearly imaged at high resolution, and the former could be classified according to the degree of PTA staining. In islets of Langerhans, the microvascular system used as an outlet by the secretory cells was also clearly observed. Microvascular system is also critically involved in the onset of diabetic complications and was clearly visible in subcutaneous tissue imaged by ASEM. The results suggest the use of in-solution ASEM for histology and to study vesicle secretion systems. Further, the high-throughput of ASEM makes it a potential tool for the diagnosis of exocrine and endocrine-related diseases.