Based on the information presented, how does aldosterone most likely enter target cells

Smooth Endoplasmic Reticulum

sER is a membrane-bound network of tubules (see Figs. 1-1 and 1-3) without surface ribosomes. sER is not involved in protein synthesis. Its main function is the synthesis of lipids, steroids, and carbohydrates, as well as the metabolism of exogenous substances, such as drugs or toxins. Cells, such as hepatocytes, that are important for synthesis of lipids and metabolism of drugs or toxins have abundant sER, as do cells that produce steroid hormones, such as adrenocortical cells and certain testicular or ovarian cells. Cells with abundant sER have pale eosinophilic, finely vacuolated cytoplasm.

Localization of Organelles

Smooth endoplasmic reticulum (SER) is an organelle that is likely to be involved in sequestering calcium. Depending on the particular brain regions, few, many, or most of the dendritic spines contain SER (Figure 1). For example, only about 14% of the hippocampal CA1 spines contain SER, and most of it occurs laminated with dense-staining material into a structure known as the spine apparatus (as in Figure 1) in the large, complex spines. In contrast, nearly 100% of the cerebellar Purkinje spines contain SER in a tubular network. Polyribosomes are also present in or near the base of some dendritic spines (Figure 2(d)), suggesting that local protein synthesis of dendritic mRNAs can occur in the spines. In immature neurons, synaptic activity can shift the distribution of polyribosomes from the shaft to spines. Similarly, endosomal compartments including coated pits and vesicles, large vesicles, tubules, and multivesicular bodies are restricted to a subpopulation of dendritic spines that differs from spines that contain SER. Mitochondria rarely occur in dendritic spines and are usually restricted to those that are very large, complex, and highly branched. However, during periods of active synapse formation and remodeling in cultured neurons, mitochondria can localize to smaller dendritic spines.

Smooth Endoplasmic Reticulum

The smooth ER is a continuous extension of the rough ER, located more distally from the nucleus. Whereas the rough ER is shaped like flattened hollow pancakes in many cell types, the smooth ER is usually more tubular in structure, forming a lacelike reticulum. It is an important site of lipid metabolism (e.g., cholesterol biosynthesis), and, for example, in liver cells, is the site where various membrane-associated detoxifying enzymes (e.g., cytochrome P450 enzymes) oxidize and otherwise act to modify toxic hydrophobic molecules (e.g., phenobarbital), making them less toxic and more water soluble.

The lumen of the smooth ER also serves as an important storage site for intracellular Ca2+. Smooth ER membranes contain ligand-regulated Ca2+ channels that open in response to the hormone-generated second messenger inositol 1,4,5-triphosphate (IP3). The cytosol of all cells is virtually Ca2+ free under resting conditions, and the transient appearance of Ca2+ in the cytosol after its release from the ER stores serves to initiate any of a number of cellular responses to extracellular signals, depending on the cell type. The ER membrane also possesses numerous Ca2+ pumps that bring the transiently released Ca2+ back into the ER lumen. Muscle contraction is initiated by transient release of Ca2+ from a specialized form of smooth ER in muscle fibers, known as the sarcoplasmic reticulum.

Smooth Endoplasmic Reticulum

The SER has a variety of functions that are often more prominent in certain cell types whose roles require an enhanced SER ability. Four common functions are the mobilization of glucose from glycogen, calcium storage, drug detoxification, and the synthesis of lipids. Glucose is stored as the polymer glycogen in close proximity to the SER, especially in liver, kidney, and intestinal cells that specialize in glucose homeostasis. To be released for use, individual glucose units are excised from glycogen and converted to glucose-1-phosphate, which is then converted to glucose-6-phosphate. However, to be exported from the cell for use by other cells, the glucose must traverse the plasma membrane; to do this, the phosphate must be removed. The enzyme that removes phosphate, glucose-6-phosphatase, is an SER-bound protein, prominent in liver, kidney, and intestine, which are organs that are glucose reservoirs. Type 1 glycogen storage disease (Von Gierke disease), one of about a dozen diseases that affect glycogen metabolism, is due to a genetic deficiency of glucose-6-phosphatase. Patients with this disease can store glycogen but cannot break it down, and with time glycogen accumulates, enlarging the liver. The disease causes chronic low blood sugar, abnormal growth, and is frequently fatal.

The SER is a storage site for calcium within cells. Calcium is pumped into the SER by active transport and released in response to hormonal signals. This is particularly important in muscle cells where the SER is so prominent it has a special name, the sarcoplasmic reticulum. Calcium is released in response to signaling pathways initiated on neurotransmitter binding to the cell-surface receptors.

Cytochrome P450s are a large family of enzymes resident in the membrane of the SER that use oxygen and nicotinamide adenine dinucleotide phosphate (NADPH) to hydroxylate a wide variety of substrates, including steroids and drugs. Hydroxylation often increases the solubility of hydrophobic drugs, facilitating clearance from the body, and selected cytochrome P450 enzymes are up-regulated in response to different drugs. This up-regulation can be large enough to cause dramatic expansion of the SER membrane. For example, chronic barbiturate use leads to expansion of the SER caused by induction of detoxifying cytochrome P450 enzymes. The increased inactivation of the drug requires larger barbiturate doses to achieve an effect, which is part of the addictive spiral in chronic users. Carcinogens, such as polycyclic aryl hydrocarbons, are also hydroxylated by SER-associated cytochrome P450 enzymes, which frequently enhances their carcinogenic activity.

Phospholipids, ceramide, and sterols are primarily synthesized in mammalian cells by enzymes in the ER, usually associated with the cytoplasmic leaflet of the SER. Exceptions to this include mitochondria that make selected phospholipids and peroxisomes that can biosynthesize cholesterol and some other lipids. The initial step in phospholipid synthesis is the condensation of two molecules of fatty acyl coenzyme A (CoA) with glycerol phosphate to make phosphatidic acid (Fig. 4-5). Each molecule of fatty acyl CoA is added separately, enabling the cell to control the type of fatty acid esterified to the 2 and 3 positions of the glycerol, with position 2 often containing an unsaturated fatty acid. Free fatty acids in the cytosol are usually bound to a fatty acid–binding protein and are converted to the fatty acyl-CoA derivatives that are substrates for the acyl-transferase enzymes in the cytosolic side of the membrane. A phosphatase removes the phosphate from phosphatidic acid to make diacyl glycerol, and in the polar head group, either cytidine-diphosphoethanolamine (CDP-ethanolamine) or cytidine-diphosphocholine (CDP-choline) is added (see Fig. 4-5).

Phospholipids are assembled in the cytoplasmic leaflet of the ER and must then be translocated to the other half of the bilayer to distribute a particular phospholipid between the two monolayers. The spontaneous flipping of phospholipids from one monolayer to the other is extremely slow and proteins termed flippases have evolved that catalyze the flipping of specific lipids. Phospholipids are often asymmetrically distributed in the two halves of a bilayer; for example, phosphatidylcholine and sphingomyelin are predominantly in the luminal face (or topologically equivalent extracellular face) of the membrane, whereas phosphatidylethanolamine and phosphatidylserine are mainly on the cytosolic face. Because the distribution of a phospholipid on the two sides of the membrane depends on the type of flippase present, it is believed that the asymmetric distribution of phospholipids in the two halves of a membrane is achieved by control of flipping, although it is unclear how the process is regulated to achieve the diversity of lipid asymmetry that is observed with different membranes.

Ceramide, the precursor of phosphosphingolipids and glycosphingolipids, is synthesized in the ER from serine and palmitoyl CoA. Phosphosphingolipids are also made in the ER. Glycosphingolipids, such as gangliosides, are made when ceramide reaches the Golgi complex and is glycosylated on the luminal face of the Golgi complex by glycosyl transferases. Glycosphingolipids are found only on the extracellular (luminal) side of membranes, suggesting that there are no flippases for this type of lipid.

The committed step in cholesterol synthesis, the production of mevalonate, is catalyzed by 3-hydroxy-3-methlyglutaryl-CoA reductase (HMG-CoA reductase), an integral membrane proteins of the SER. Other enzymes involved in the process of making cholesterol, as well as metabolically modifying cholesterol, are also ER residents. Although initially made on the cytosolic side of the SER, cholesterol is found on both sides of the membrane, and evidence exists that cholesterol flippases catalyze the flipping.

Not only are many lipids unevenly arranged in two halves of the bilayer, but most membranes maintain a unique lipid composition. For example, the ER of mammalian cells is typically 50% or more phosphatidylcholine, with less than 10% each for sphingomyelin and cholesterol, whereas the plasma membrane contains less than 25% phosphatidylcholine and more than 20% each for sphingomyelin and cholesterol. Thus, once a lipid is incorporated into the ER, it must not only be transported to other membranes, but the characteristic compositions of the destination membranes must be maintained. There are three basic thought processes on how this occurs. One is vesicle-mediated transport whereby vesicles bud from the donor membrane and fuse to a target membrane, thus moving lipids from one membrane to the other. Known pathways of vesicular transport whereby vesicles originating from the ER move sequentially through the Golgi apparatus and on to endosomes or the plasma membrane have been reported (see later). Ceramides that require glycosylation in the Golgi complex to make glycosphingolipids are believed to use this pathway. Cholesterol, however, can go from the ER to other membranes bypassing the Golgi complex, and evidence exists for a type of vesicle that buds from the ER carrying cholesterol and selected phospholipids that transports lipids to other membranes without passing through the Golgi complex. A second idea in lipid transport is that lipid transfer proteins directly extract a lipid from a membrane and shield the lipid in a hydrophobic pocket while the protein diffuses through the cytosol and deposits the lipid in an acceptor membrane. The third idea in lipid transport is that the ER makes transient contact with another membrane, passing lipids from the ER to the other membrane.

Hepatotoxicity secondary to enzyme induction

Proliferation of smooth endoplasmic reticulum (SER) is a well-documented change seen secondary to administration of xenobiotics that induce cytochrome P450 enzymes (Guengrich, 2007). Hepatocytes have a full complement of cytochrome P450s (CYP), since one of the liver’s main functions is metabolism of foreign substances. Induction most commonly occurs in centrilobular hepatocytes. Not all xenobiotics that induce cytochrome P450s cause proliferation of smooth endoplasmic reticulum. Proliferation is dependent on the subset of CYP that are induced, as no single agent induces all CYP. Cytochrome P450 enzymes are grouped into families CYP1, CYP2, CYP3, etc., with capital letters designating subfamilies CYP1A, CYP1B, CYP1C, etc., and numerals further identifying individual enzymes (e.g. CYP1A1). Not all CYP inducers cause SER proliferation and liver enlargement (Barka and Popper, 1967); however, SER proliferation is nearly pathognomonic for CYP induction.

Classic inducers of SER proliferation are phenobarbital and its analogs. Phenobarbital is metabolized by, and induces, cytochrome P4502B, the major membrane protein of SER (2d). Proliferation can be rapid and dramatic, with morphologic changes occurring within 4 days. Liver weight and size are increased. By light microscopy in HE sections, centrilobular hepatocytes are hypertrophied due to increased amounts of homogeneous eosinophilic cytoplasm. SER proliferation begins in centrilobular hepatocytes (Figure 54.5). With increasing magnitude, proliferation will extend into midzonal and rarely to periportal hepatocytes. Withdrawal of the inciting xenobiotic is accompanied by a rapid regression of changes (Cheville, 1994). Effete SER forms aggregates and is removed from the cell by autophagocytosis (Feldman et al., 1980).

Hepatotoxicity secondary to hepatocellular enzyme induction can occur through increased activation of xenobiotics to hepatotoxins (Zimmerman, 1999). Subsets of CYP inactivate xenobiotics while others activate to reactive electrophiles (Greaves, 2007). Aflatoxin B1, a mycotoxin, is activated to a number of metabolites, including exo-8,9-epoxide, an hepatocarcinogen. Acetaminophen, an analgesic, is activated to a reactive iminoquinone. Trichloroethylene (TCE), an industrial toxicant and previously used anesthetic, is activated to TCE oxide, which forms unstable protein adducts. Troglitazone, an antidiabetes drug, is metabolized to electrophilic reactive metabolites.

SERCA, another P-type Ca pump, was first identified in sarcoplasmic reticulum

There are three isoforms of SERCA that are products of separate genes. SERCA-1 is expressed in fast-twitch skeletal muscle; SERCA-2a in cardiac/slow-twitch muscle; SERCA-2b, an alternatively spliced form, is expressed in smooth muscle and non-muscle tissues; and SERCA-3 is expressed in endothelial, epithelial, and lymphocytic cells and platelets. SERCA-2b is the major form expressed in brain, where it is found predominantly in neurons. SERCA pumps Ca2+ from cytosol into the ER for storage. Ca2+ is released from the ER through the IP3 receptor (IP3R) when it binds the signal molecule IP3. The Ca pumps of ER normally reduce cytoplasmic [Ca2+] to <1 mM. However, rapid restoration of such low [Ca2+] subsequent to plasma membrane depolarizations requires coordinate activity of the plasmalemma Na,Ca antiporter (see discussion below).

1. Endoplasmic Reticulum

Many compounds cause an increase in smooth endoplasmic reticulum (SER) or cytochrome P450 monooxygenase activities. Phenobarbital and 3-methylcholanthrene are considered model compounds for enzyme induction. Phenobarbital causes an increase in a wide variety of monooxygenase activities and may be associated with a two- to five fold increase in SER. This leads to the appearance of centrilobular hypertrophy with amorphous eosinophilic hepatocytic cytoplasm (Fig. 12B). Although the increase is mainly due to the additional production of SER, part of the SER increase may be related to impaired membrane catabolism. Compounds such as 3-methylcholanthrene cause increases in only a few specific monooxygenase activities and may not show morphologic evidence of SER increase. trans-Stilbene oxide has an intermediate effect: it induces several of the enzymes that phenobarbital does, with less extensive increases in SER. SER changes and associated enzyme induction are reversible following the withdrawal of chemical exposure.

The mechanism of cytochrome P450 induction and SER increase by barbiturates remains obscure. Response elements in promter-enhancer regions of CYP2B genes have been found. However, the experimental identification and confirmation of positively (or negatively) acting proteins that serve as receptors for mediating this response remains largely incomplete. A receptor designated CAR (constitutively activated receptor), a member of the steroid/thyroid receptor superfamily, has been linked to phenobarbital activation of gene transcription by the promoter region of the CYP2B gene.

A Corticosteroidogenesis

At the membranes of the smooth endoplasmic reticulum, pregnenolone can be metabolized along either the 17α-hydroxysteroid pathway or the 17-deoxysteroid pathway. The former pathway leads to 11-deoxycortisol and is accomplished by the actions of the enzyme 21-hydroxylase (P450c21). 11-Deoxycortisol may be secreted or may reenter the mitochondria, where the 11β-hydroxylase enzyme (P45011β1) converts it to cortisol. Conversion of pregnenolone to progesterone in the zona fasciculata or glomerulosa via the 17-deoxysteroid pathway yields 11-deoxycorticosterone that is converted to corticosterone by employing the same enzymes as did the17α-hydroxysteroid pathway. Cortisol and/or corticosterone are the major end products of corticosteroidogenesis in the zona fasciculata and to a lesser extent in the zona reticularis.

In the zona glomerulosa, the 17-deoxysteroid pathway is favored, and corticosterone is further modified to 18-hydroxycorticosterone and then to aldosterone. Another mitochondrial enzyme called aldosterone synthase (P45011β2 or P450aldo) is responsible for aldosterone synthesis in rodents and humans. Aldosterone and 18-hydroxycorticosterone have mineralocorticoid activity, but aldosterone is more potent and is typically the dominant secretory product of the zona glomerulosa in vivo. However, deoxycorticosterone may be the major secretory product in some species (see Table 8.1).

As mentioned previously, adrenal androgens are synthesized primarily in the zona reticularis or by the fetal zone of primates. Both the Δ4 and Δ5 pathways may be utilized (see Chapter 3, Fig. 3.22), but the Δ5 pathway is more common, resulting in DHEA that is then sulfated to form DHEA-S. The primate fetal zone makes little or no androstenedione, and this weak Δ5 androgen is more commonly found in the adult adrenal. DHEA also is a weak androgen, and the sulfated form does not penetrate readily into potential target cells, reducing the threat of masculinization to a female fetus or the mother. DHEA-S is converted by the placenta into 16α-hydroxy-DHEA and then by aromatase (P450aro) to estriol. Androstenedione is converted by P450aro to estrone, which can be processed further to estradiol or estriol. These conversions of fetal androgens are essential for maintaining pregnancy. Because the primate placenta cannot synthesize androgens, it must depend entirely on the fetal adrenal to provide the androgen precursor for estrogen synthesis.

Endoplasmic Reticulum, Ribosomes, and Golgi Apparatus

Rough endoplasmic reticulum (RER), smooth endoplasmic reticulum (SER), and Golgi complexes are abundant in mammalian hepatocytes (see Figs. 1-15 and 1-16).1,2,51,58 Their functions are related mainly to the synthesis and conjugation of proteins, metabolism of lipids and steroids, detoxification and metabolism of drugs, and breakdown of glycogen. The endoplasmic reticulum forms a continuous three-dimensional network of tubules, vesicles, and lamellae. Almost 60% of the endoplasmic reticulum has ribosomes attached to its cytoplasmic surface and is known as the RER. The remaining 40% constitutes the SER, which lacks a coating of ribosomes. The membranes of the endoplasmic reticulum are between 5 nm and 8 nm thick. The lumen of the RER is approximately 20 nm to 30 nm wide, whereas that of the SER is larger (30-60 nm). The morphologic characteristics and amount of the endoplasmic reticulum vary in the different zones of the liver lobule.

RER is arranged in aggregates of flat cisternae that may be found throughout the cytoplasm. It is more frequently distributed in the perinuclear, pericanalicular, and subbasilar regions of hepatocytes, and it is more abundant in periportal cells than in centrilobular cells.59 The numerous attached membrane-bound ribosomes consist of a large and a small subunit, with the large subunit attached to the RER. Free ribosomes and polyribosomes are also present within the hepatocyte cytoplasm. Ribosomes contain RNA and ribosomal proteins and play a key role in the synthesis of proteins, particularly those destined for secretion or for delivery to intracellular membrane compartments or the plasma membrane. Vesicles containing these proteins are directed to the proximate (cis) cisternae of the Golgi apparatus, for further processing.

SER is less common and has a more complex arrangement than RER.51 It is usually much more abundant in centrilobular than in periportal hepatocytes59,60; the high content of heme-containing cytochromes lends a darker pigmentation to the centrilobular region of the lobule, as is evident on visual inspection of the cut surface of the liver. The matrix within the SER tubules is usually slightly more electron-dense than the surrounding cytoplasm. SER membranes are irregular in size and present a tortuous course. They may be tubular or vesicular in structure with a width of 20 nm to 40 nm. SER is mainly distributed near the periphery of the cell. It is often in close relation to RER and Golgi membranes, as well as to glycogen inclusions.51

The ER is not the only site of protein synthesis in hepatocytes. Abundant free ribosomes in the cytoplasm participate in the synthesis of some proteins that will be secreted but synthesize essentially all of the structural proteins for the hepatocyte. Proteins that are to remain within the cytoplasm or are destined to enter the nucleus, peroxisomes, or mitochondria are completely synthesized by free ribosomes.

The Golgi complex is a three-dimensional structure in hepatocytes, characteristically consisting of a stack of four to six parallel cisternae, often with dilated bulbous ends containing electron-dense material.10,51,58 Multiple Golgi complexes exist in each hepatic parenchymal cell, generally distributed near the nucleus. This structure shows a convex or proximal portion facing the nucleus and the endoplasmic reticulum (cis-Golgi), where small vesicles transfer proteins from the endoplasmic reticulum to the Golgi, and a concave part (trans-Golgi), which connects with a post-Golgi trans-Golgi network that directs proteins towards their final destinations: to organelle membranes of the cell, the plasma membrane, or for secretion. The cisternae may be up to 1 µm in diameter with a lumen that is 30 nm wide. The Golgi complex is capable of rapid and reversible structural reorganization into a tubuloglomerular network, while maintaining its biosynthetic capabilities.61 With the SER, RER, lysosomes, other intermediate organelle compartments, and even the nuclear and mitochondrial envelope membranes, the Golgi is an integral part of the complex intracellular organelle network involving vesicular trafficking that enables uptake, sorting, degradation, biosynthesis, trafficking, and/or secretion of cellular proteins and lipids.60-63