Bacterial lipoteichoic acid induces capsular contracture by activating innate immune response.

BACKGROUND: Capsular contracture is attributed to an exaggerated fibrosis response within the capsule and is partly associated with bacterial contamination in situ. However, the cellular mechanisms that initiate this response are unclear. METHODS: We developed a mouse model of capsular contracture by repeated injection of 10 µg/ml lipoteichoic acid (LTA). The histological changes in the capsule tissue were measured by hematoxylin-eosin, Masson, and immunohistochemical staining. The expression of cytokines was measured by quantitative reverse-transcription polymerase chain reaction. We also used pharmacological methods to verify the roles of macrophages and Toll-like receptor 2 (TLR2) signaling in this pathological process. RESULTS: We discovered that repeated LTA injection, at a low concentration, could induce the thickening of the capsule tissue. Macrophage infiltration and TLR2/nuclear factor-kappa B (NF-κB) signaling activated in this process could be suppressed by macrophage depletion or TLR2 receptor inhibition. CONCLUSIONS: As TLR2 signal activation was found to cause capsular contracture by inducing macrophage infiltration as a consequence of trace amounts of LTA contamination in situ, this target is helpful for understanding that chronic or repeated subclinical infection could activate capsular contracture.

Antiphotoaging Effect of Micronized Fat in Ultraviolet B-Induced Human Dermal Fibroblasts.

BACKGROUND: Adipose-derived stromal vascular fraction (SVF) and mesenchymal stem cells have been proven to reduce the effects of skin photoaging. However, there is no standardized protocol for their preparation. This study aimed to investigate the skin rejuvenation potential of micronized fat, obtained using a novel device attached with a trifoliate blade, in the ultraviolet B (UV-B)-induced human dermal fibroblast model. METHODS: Micronized fat was prepared to obtain adipose-derived SVF, and the adipose-derived mesenchymal stem cell-to-SVF ratio was determined by flow cytometry. The UV-B-induced human dermal fibroblasts model was constructed to identify the characteristics of the human dermal fibroblasts using vimentin and S-100 immunostaining, observe their morphology, and measure the levels of photoaging-related factors. After the previous steps were completed, different cell groups were co-cultured with UV-B-induced human dermal fibroblasts, and the extent of improvement of photoaging was evaluated. RESULTS: Micronized fat had a higher adipose-derived mesenchymal stem cell-to-SVF ratio than the control fat preparations. The UV-B-induced human dermal fibroblasts model showed lowered levels of type I collagen and transforming growth factor-ß and increased expression of matrix metalloproteinases (MMPs), which are the characteristics of photoaging in normal human dermal fibroblasts. Compared with different cell groups co-cultured with UV-B-induced human dermal fibroblasts, micronized fat could lower the expression of MMPs and increase the level of type I collagen but lower the level of transforming growth factor-ß. CONCLUSIONS: Obtaining micronized fat is more effortless and clinically safer. Micronized fat has an antiphotoaging effect by inhibiting the expression of MMPs by means of the mitogen-activated protein kinases signaling pathway. CLINICAL RELEVANCE STATEMENT: The authors' work has potential clinical applications in fat grafting for facial rejuvenation.

Repeated Lipoteichoic Acid Injection at Low Concentration Induces Capsular Contracture by Activating Adaptive Immune Response through the IL-6/STAT3 Signaling Pathway.

BACKGROUND: Capsular contracture is the most common complication of breast implantation surgery. Bacterial contamination was considered to play an important role in the occurrence of capsular contracture, and Gram-positive bacteria such as Staphylococcus epidermidis were discovered in the clinical specimens. Lipoteichoic acid (LTA) was a component of the cell wall of Gram-positive bacteria and was sufficient in the pathogenicity of the bacteria. The authors assumed that LTA could trigger the immunologic response against the implant and cause capsular contracture. METHODS: The authors developed a rat model of capsular contracture by repeated injection of 10 µg/mL LTA. The histologic changes of the capsule tissue were measured by hematoxylin and eosin, sirius red, Masson, and immunohistochemical staining. The expression of related cytokines was measured by quantitative real-time polymerase chain reaction. The downstream pathway activation was shown by Western blot. The authors also applied tocilizumab, an interleukin (IL)-6 receptor antagonist, to verify the role of IL-6 in this pathologic process. RESULTS: The authors discovered that repeated LTA injection, at a low concentration, could induce the thickening of capsule tissue, the deposition of collagen fiber, and the activation of myofibroblasts. The IL-6/signal transducer and activator of transcription 3 signaling pathway was activated in this process, and the inhibition of IL-6 receptor could relieve the symptoms. B cells and T-helper cells, especially T-helper type 1, could be related to this phenomenon. CONCLUSIONS: The authors' research corroborated that subclinical infection could trigger capsular contracture, and the immune system played an important role in this process. The authors' results provided a possible research direction for the mechanism of bacterial infection-induced immune response against breast implants. CLINICAL RELEVANCE STATEMENT: The authors' research provides a possible research direction for the mechanism of bacterial infection-induced immune response against breast implants, and a potential target for predicting the prognosis of capsular contracture.

Denervation Affected Skin Wound Healing in a Modified Rat Model.

Introduction: Lacking of normal innervation increases the chance of chronic wounds and recurrence of ulceration. Various rodent models are designed to reveal nerve-wound relationship but present many limitations to mimic human wound which heals primarily by re-epithelialization rather than contraction in rodents. This article tested a modified rat model of denervated wound healing to better mimic clinical common denervated wounds. Material and Methods: The wounds formed on right hind paws of 18 SD rats served as the experimental (denervated) group and the left side as contra-lateral control (non-denervated). The denervation was achieved through sciatic and femoral nerve co-transection and the control side underwent sham-surgery 3 days prior to a skin punch wound formation on both sides. Wound closure rate was calculated under digital photographing. Loss of innervation and affected healing process was confirmed by histological analyses. Results: Truncation of the sciatic and femur nerve successfully denervated the skin of the hind paw and resulted in a significantly declined healing rate, prolonged inflammation, weakened dermal contraction, hindered macrophage recruitment, retarded re-epithelialization and collagen deposition, decreased angiogenesis and epidermal proliferation, and persisted epidermal apoptosis compared to the innervated contra-lateral control. Conclusion: Wound on denervated dorsal pedis in rats can be used to study denervated skin healing in multiple histological process. We believe that this model will assist in understanding the underlying mechanism of nerve-wound relationship and identifying new treatment strategies that can be more rapidly translated into clinical practice.

PSMC2 knockdown suppressed tumor progression of skin cutaneous melanoma.

Skin cutaneous melanoma (SKCM) is the most lethal tumor among three of the major malignant cancers of the skin. The mechanism underlying the malignant biological behaviors of SKCM is not fully clear. Our study intended to verify the molecular mechanism of proteasome 26 S subunit ATPase 2 (PSMC2) in malignant biological behaviors of SKCM. The Cancer Genome Atlas (TCGA) database was used to analyze the expression of PSMC2 in SKCM and its impact on prognosis. PSMC2 expression in 105 paired SKCM tissues was investigated by immunohistochemistry (IHC), its functional roles were verified using a series of cell experiments, and the underlying pathway was detected by protein-chip technology and gene set enrichment analysis. We found that PSMC2 was significantly upregulated in SKCN patients from TCGA datasets and verified in clinical SKCM tissues. Moreover, high PSMC2 was shown to closely correlate with the pathological stages and lymphatic metastasis of SKCM patients. Functionally, knockdown of PSMC2 suppressed the progression of SKCM through inhibiting cell proliferation, migration, and DNA damage in vitro as well as cell growth in vivo, whereas inducing apoptosis, cycle arrest in G2 phase. Similarly, pharmaceutical inhibition of proteasome with MG132 mimicked the PSMC2 knockdown induced defects in cell cycle arrest, apoptosis and proliferation, while overexpression of PSMC2 has the opposite effects. Mechanistically, the silence of PSMC2 remarkably elevated the pro-apoptotic proteins DR6, IGFBP-4, p21, and p53, while inhibited the anti-apoptosis protein TRAILR-3 and the proteins related to the Wnt signaling pathway. The present study revealed that PSMC2 participated in a positive regulation to promote the progression of SKCM through regulating the Wnt signaling pathway. Our findings may offer a new mechanism underlying the development and progression of SKCM, and a deeper understanding of PSMC2 may contribute to SKCM treatment.

Kindlin-2 regulates the differentiation of 3T3-L1 preadipocytes: implications for wound healing.

BACKGROUND: Adipose tissue has been proven to play a crucial role in wound healing, while kindlin-2, an integrin-associated protein, has been shown to regulate cell adhesion, migration, and differentiation. This study aimed to explore its involvement in the cell differentiation of 3T3-L1 preadipocytes and its role in wound healing. METHODS: Cell adhesion, Cell Counting Kit-8 (CCK-8), Transwell, and in vitro wound healing assays, along with adipogenic and osteogenic differentiation induction were performed in 3T3-L1 preadipocytes in which kindlin-2 was knocked down or overexpressed. In vivo, kindlin-2 (+/-) transgenic mice were constructed, and wound healing was analyzed by immunohistochemistry (IHC) in a mouse dorsal wound model. Real-time polymerase chain reaction (RT-PCR) and western blotting were performed to analyze the expression of adipokines and adipogenic markers in mouse wound tissues. Adipogenic differentiation induction of adipose tissue stromal vascular fraction (SVF) were performed, and the expression of adipogenic markers in SVF was detected by western blotting. The target signaling pathway highly related to adipogenic differentiation was explored by computational biology and verified by western blotting. RESULTS: Knockdown of kindlin-2 was found to inhibit the adhesion, migration, and adipogenic differentiation of 3T3-L1 preadipocytes while promoting their osteogenic differentiation. In contrast, kindlin-2 overexpression resulted in increased adhesion, migration, and adipogenic differentiation of 3T3-L1 preadipocytes while reducing osteogenic differentiation. In vivo, downregulation of kindlin-2 inhibited adipogenesis in kindlin-2 transgenic mice, resulting in delayed wound healing by inhibiting inflammation, angiogenesis, collagen remodeling, and wound contraction. Mechanistically, we found that kindlin-2 could regulate adipogenic differentiation through PI3K/AKT/mTOR signaling pathway. CONCLUSIONS: Our study revealed the essential role that kindlin-2 has in the differentiation and wound healing of 3T3-L1 preadipocytes, which offers a theoretical basis for further research and a novel strategy for wound healing.

Protective Effects of Micronized Fat against Ultraviolet B-Induced Photoaging.

BACKGROUND: Autologous fat grafting has long been an essential technique in cosmetic and reconstructive surgery. Here, the authors report the advantages of a new device for preparing micronized fat, and they also investigated the therapeutic potential of micronized fat against ultraviolet B-induced photoaging. METHODS: Micronized fat aliquots were prepared through a connector device with trifoliate blades. The histologic structure and viability of the prepared fat samples were evaluated by calcein AM/propidium iodide staining. The levels of growth factor were measured by enzyme-linked immunosorbent assay, and flow cytometry was used to detect the ratio of adipose-derived mesenchymal stem cells to stromal vascular fraction. The authors also evaluated the effects of micronized fat transplantation through immunohistochemistry and Masson trichrome staining in an animal model of photoaging. RESULTS: The micronized fat had a normal histologic structure and viable adipocytes. It had a higher level of hepatocyte growth factor compared with the control group, and its ratio of adipose-derived mesenchymal stem cells to stromal vascular fraction was also higher than in the control fat preparations. Transplantation of micronized fat preparations in the animal model of photoaging produced increased skin neovascularization, epidermal cell proliferation, and dermal collagen density. CONCLUSIONS: The authors' results demonstrated that the novel device produced micronized fat easily, which can condense adipose tissue. This micronized fat was easy to use with smaller cannulas. It mitigated the signs of cutaneous photoaging and was superior to control fat. Contrary to previous reports, normal histologic structures and viable adipocytes were noted in the micronized fat.

Elevated kindlin-2 promotes tumour progression and angiogenesis through the mTOR/VEGFA pathway in melanoma.

BACKGROUND: In our previous study, kindlin-2 promoted skin wound healing and decreased the permeability of neovascularization during angiogenesis. Herein, we explored the biological function and underlying mechanism of kindlin-2 in cutaneous melanoma. METHODS AND RESULTS: Through a series of in vitro assays, we found that high levels of kindlin-2 promoted migration and invasion of melanoma cells without influencing cell proliferation. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analyses showed that upregulated kindlin-2 promoted the cellular epithelial-mesenchymal transition (EMT). Importantly, we found that melanoma cells overexpressing kindlin-2 promoted angiogenesis and VEGFA secretion in vitro and facilitated tumour growth and lung metastasis in vivo. To unveil the underlying mechanism, we conducted Next-generation sequencing (NGS) and differential expression analyses. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that overlapping differentially expressed genes (DEGs) were primarily enriched in the TGF-ß, mTOR and VEGF signalling pathways. Then, we confirmed that the mTOR/VEGFA pathway was activated during the process of kindlin-2-induced melanoma progression and angiogenesis. Moreover, we demonstrated that kindlin-2 was significantly overexpressed in clinical melanoma samples and that a high level of kindlin-2 predicted a poor prognosis. CONCLUSIONS: Taken together, these findings showed that kindlin-2 promotes angiogenesis and tumour progression via the mTOR/VEGFA pathway.

Polydopamine-mediated covalent functionalization of collagen on a titanium alloy to promote biocompatibility with soft tissues.

The clinical success of a titanium (Ti) percutaneous implant requires the integration with soft tissues to form a biological seal, which effectively combats marsupialization, premigration and infection after implantation. However, the bioinert surface of Ti or its alloys prevents the material from sufficient biological sealing and limits the application of Ti or its alloys as percutaneous implants. In this study, we achieved a collagen coating to bioactivate the surface of Ti-6Al-4V. In order to enable covalent functionalization, we first deposited a polydopamine (PDA) coating on Ti-6Al-4V based on dopamine self-polymerization and then immobilized collagen chains on PDA. Compared with physical absorption, such a chemical bonding method through mussel-inspired chemistry showed better stability of the coating. Meanwhile, the cellular tests in vitro indicated that collagen functionalization on the Ti-6Al-4V surface showed better adhesion of human foreskin fibroblasts (HFFs) and human immortal keratinocytes (HaCaTs). The subcutaneous implantation tests in rats indicated that the collagen modification attenuated soft tissue response and improved tissue compatibility compared with either pure Ti-6Al-4V or merely PDA coated samples. The facile bioinspired approach enables a persistent modification of metals by macromolecules under aqueous environments, and the PDA-collagen coated titanium alloy is worthy of further investigation as a percutaneous implant.

Knockdown of the KINDLIN-2 Gene and Reduced Expression of Kindlin-2 Affects Vascular Permeability in Angiogenesis in a Mouse Model of Wound Healing.

BACKGROUND Angiogenesis is an important component of wound healing and tissue repair. Kindlin-2 is an integrin-associated protein, encoded by the KINDLIN-2 gene, which has been shown to affect cell adhesion and migration of cells, including endothelial cells. The aim of this study was to use a mouse model of wound healing to evaluate the effects of expression of KINDLIN-2 on angiogenesis in wound healing in vivo. MATERIAL AND METHODS Thirty-six male C57BL/6 mice were studied in an established model that used a wound created on the back. Mice were divided randomly into three groups: the normal group (n=12) received injections of normal (0.9%) saline; the KINDLIN-2(-) group (n=12) received injections of adeno-associated virus with small interfering (si)RNA targeting the KINDLIN-2 gene (AAV-KINDLIN-2-siRNA); and the control (group (n=12) received injections of adeno-associated virus containing a scrambled RNA sequence (AAV-control-RNA). Wound healing was analyzed by biochemical examination of the exudates and histology. Evans blue dye was injected into the caudal vein of each mouse, two weeks after wound healing to assess neovascular permeability. RESULTS Wound healing was significantly delayed in the KINDLIN-2 gene knockdown mice (AAV-KINDLIN-2-siRNA) compared with that of the normal group and the control group, and neovascular permeability was increased. In the AAV-KINDLIN-2-siRNA group, blood vessels were shorter and thinner compared with the normal group and the control group. CONCLUSIONS In a mouse model of wound healing, KINDLIN-2 gene knockdown inhibited wound healing, and increased neovascular permeability in vivo.

HDAC6 interacts with PTPN1 to enhance melanoma cells progression.

Histone deacetylase 6 (HDAC6) plays an important role in oncogenic transformation and cancer metastasis. Our previous study has demonstrated that HDAC6 was highly expressed in melanoma cells, and contributed to the proliferation and metastasis of melanoma cells. However, the underlying mechanism of HDAC6 in melanoma metastasis and progression remains largely unclear. In this study, we reported that HDAC6 directly interacted with Tyrosine-protein phosphatase non-receptor type 1 (PTPN1) by performing co-immunoprecipitation (Co-IP) combined with liquid chromatography tandem mass spectrometry (LC-MS/MS). HDAC6 increased the protein level of PTPN1 independent of histone modifying activity. In addition, PTPN1 promoted proliferation, colony formation and migration while decreased apoptosis of melanoma cells through activating extracellular signal-regulated kinase 1/2 (ERK1/2). Furthermore, we found that matrix metallopeptidase 9 (MMP9) was increased by HDAC6/PTPN1/ERK1/2 axis, which might serve as a mechanism for melanoma invasion and metastasis. In conclusion, HDAC6 might enhance aggressive melanoma cells progression via interacting with PTPN1, which was independent of its histone modifying activity.

Memory retrieval in addiction: a role for miR-105-mediated regulation of D1 receptors in mPFC neurons projecting to the basolateral amygdala.

BACKGROUND: Drug addiction is a chronic brain disorder characterized by the compulsive use of drugs. The study of chronic morphine-induced adaptation in the brain and its functional significance is of importance to understand the mechanism of morphine addiction. Previous studies have found a number of chronic morphine-induced adaptive changes at molecular levels in the brain. A study from our lab showed that chronic morphine-induced increases in the expression of D1 receptors at presynaptic terminals coming from other structures to the basolateral amygdala (BLA) played an important role in environmental cue-induced retrieval of morphine withdrawal memory. However, the neurocircuitry where the increased D1 receptors are located and how chronic morphine increases D1 receptor expression in specific neurocircuits remain to be elucidated. RESULTS: Our results show that chronic morphine induces a persistent increase in D1 receptor expression in glutamatergic terminals of projection neurons from the medial prefrontal cortex (mPFC) to the BLA, but has no influence on D1 receptor expression in projection neurons from the hippocampus or the thalamus to the BLA. This adaptation to chronic morphine is mediated by reduced expression of miR-105 in the mPFC, which results in enhanced D1 receptor expression in glutamatergic terminals of projection neurons from the mPFC to the BLA. Ex vivo optogenetic experiments show that a chronic morphine-induced increase in D1 receptor expression in glutamatergic terminals of projection neurons from the mPFC to the BLA results in sensitization of the effect of D1 receptor agonist on presynaptic glutamate release. mPFC to BLA projection neurons are activated by withdrawal-associated environmental cues in morphine-withdrawal rats, and overexpression of miR-105 in the mPFC leads to reduced D1 receptor induction in response to chronic morphine in glutamatergic terminals of the projection neurons from the mPFC to the BLA, and a reduction in place aversion conditioned by morphine withdrawal. CONCLUSIONS: These results suggest that chronic morphine use induces a persistent increase in D1 receptors in glutamatergic terminals of projection neurons from the mPFC to the BLA via downregulation of miR-105 in the mPFC, and that these adaptive changes contribute to environmental cue-induced retrieval of morphine withdrawal memory.

Morphine disinhibits glutamatergic input to VTA dopamine neurons and promotes dopamine neuron excitation.

One reported mechanism for morphine activation of dopamine (DA) neurons of the ventral tegmental area (VTA) is the disinhibition model of VTA-DA neurons. Morphine inhibits GABA inhibitory neurons, which shifts the balance between inhibitory and excitatory input to VTA-DA neurons in favor of excitation and then leads to VTA-DA neuron excitation. However, it is not known whether morphine has an additional strengthening effect on excitatory input. Our results suggest that glutamatergic input to VTA-DA neurons is inhibited by GABAergic interneurons via GABAB receptors and that morphine promotes presynaptic glutamate release by removing this inhibition. We also studied the contribution of the morphine-induced disinhibitory effect on the presynaptic glutamate release to the overall excitatory effect of morphine on VTA-DA neurons and related behavior. Our results suggest that the disinhibitory action of morphine on presynaptic glutamate release might be the main mechanism for morphine-induced increase in VTA-DA neuron firing and related behaviors.

Two free radical pathways mediate chemical hypoxia-induced glutamate release in synaptosomes from the prefrontal cortex.

It has been known that the inhibition of mitochondrial cytochrome c oxidase is one of the earliest events occurring under hypoxia and this inhibition can lead to neuronal damages. Thus, the cytochrome c oxidase inhibitor sodium cyanide (NaCN) is widely used to produce a model of chemical hypoxia by inhibiting this enzyme. However, the downstream signaling pathways of the inhibition of the cytochrome c oxidase remain to be studied. In the present paper, we used sodium cyanide to mimic the inhibition of the mitochondrial cytochrome c oxidase and studied its effect on glutamate release in synaptosomes from the prefrontal cortex using on-line fluorimetry. We also further investigated the mechanisms underlying the enhancing effect of sodium cyanide on glutamate release using pharmacological approaches combined with other techniques. The results showed that sodium cyanide significantly increased glutamate release from synaptosomes of prefrontal cortex; the broad-spectrum free radical scavenger MnTBAP and melatonin completely abolished the effect of sodium cyanide on glutamate release; the H2O2-NMDA receptor pathway mediated one part, whereas the lipid peroxyl radicals-ATP synthase pathway mediated another part of the sodium cyanide-induced glutamate release; scavenging H2O2 and enhancing ATP synthase activity could completely abolish the sodium cyanide-induced glutamate release.

Chronic morphine treatment switches the effect of dopamine on excitatory synaptic transmission from inhibition to excitation in pyramidal cells of the basolateral amygdala.

Dopaminergic signaling in the basolateral amygdala (BLA) is important for drug-stimulus learning that triggers relapse to drug-seeking behavior. However, little is known about adaptive changes in this signaling pathway upon chronic morphine treatment. In this paper, we observed the influence of chronic morphine treatment on the effect of dopamine (DA) on the excitatory transmission in the pyramidal cells of BLA in slices with the whole-cell patch-clamp method. We also studied its mechanism and significance with pharmacological approaches combined with biochemical and behavioral techniques. The results showed that chronic morphine exposure switched the effect of DA on the excitatory synaptic transmission from inhibition to excitation; the chronic morphine-induced switching action on the effect of DA was due to its influence on D1 receptors; the site of the effect of chronic morphine treatment on D1 receptors was at presynaptic locus; chronic morphine treatment induced a significant increase in the amount of D1 receptor expression in the synaptosomes and synaptosomal membrane fraction from BLA; the enhancement of presynaptic glutamate release by D1 receptor agonist upon chronic morphine treatment was dependent on the activation of cAMP-dependent protein kinase; and the intra-BLA injection of D1 receptor antagonist canceled the conditioned place aversion (CPA) in morphine-dependent rats. In conclusion, chronic morphine treatment switches the effect of DA on the excitatory synaptic transmission from inhibition to excitation by the presynaptic D1 receptor amount increase-mediated glutamate release in the pyramidal cells of BLA and the blockade of D1 receptors in BLA cancels CPA in morphine-dependent rats.

Sigma-1 receptors amplify dopamine D1 receptor signaling at presynaptic sites in the prelimbic cortex.

Sigma-1 receptors are highly expressed in the brain. The downstream signaling mechanisms associated with the sigma-1 receptor activation have been shown to involve the activation of protein kinase C (PKC), the control of Ca(2) homoeostasis and the regulation of voltage- and ligand-gated ion channels. But few studies examined the regulatory effect of sigma-1 receptors on metabotropic receptor signaling. The present paper studied the regulatory effect of sigma-1 receptors on the signaling of dopamine D1 receptors, one of metabotropic receptors, by examining the effect of sigma-1 receptor agonists on the D1 receptor agonist-induced cAMP-dependent protein kinase (PKA) activation at presynaptic sites using the synaptosomes from the prelimbic cortex. The results showed that sigma-1 receptor agonists alone had no effects on the PKA activity, but could amplify the D1 receptor agonist-induced PKA activation. The sigma-1 receptor agonist also amplified the membrane-permeable analog of cAMP- and the adenylyl cyclase (AC) activator-induced PKA activation, but did not on the D1 receptor agonist-induced AC activation. The conventional PKC (cPKC), especially the PKCßI, and the extracellular Ca(2+) influx through L-type Ca(2+) channels might play key roles in the amplifying effect of the sigma-1 receptor agonists. The activation of PKC by sigma-1 receptor agonists was the upstream event of the increase in the intrasynaptosomal Ca(2+) concentration. These results suggest that sigma-1 receptors may amplify the D1 receptor agonist-induced PKA activation by sigma-1 receptors - cPKC (especially the PKCßI) - L-type Ca(2+) channels - Ca(2+) - AC and/or cAMP signaling pathway.